Aldehyde-Tagged Protein-Based Drug Carriers and Methods of Use

ABSTRACT

The disclosure provides aldehyde-tagged protein carriers that can be covalently and site-specifically bound to drug to provide a drug-containing scaffold. The invention also encompasses methods of production of such drug-containing scaffolds and intermediates, as well as methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. provisional applicationSer. No. 61/153,162, filed Feb. 17, 2009, which application isincorporated herein by reference in its entirety.

INTRODUCTION

The field of protein and small molecule therapeutics has advancedgreatly, providing a number of clinically beneficial drugs and promisingto provide more with the years to come. Protein therapeutics can provideseveral advantages in therapies, due to, for example, exquisitespecificity, multiplicity of functions and relatively low off-targetactivity, resulting in fewer side effects. With the development ofsophisticated screening methods, small molecule drugs have also advancedin specificity of action.

Often, though, such therapeutics can be further improved by providingfor enhanced activity following administration. For example, it is oftendesirable to increase the serum half-life of the therapeutic (e.g., inorder to reduce the overall dose and/or the number of administrationsrequired over a dosage period). Alternatively or in addition,therapeutics could benefit from improving their bioavailability. Forexample, some drugs may benefit from improving solubility in therelevant physiological environment and/or to facilitation formulation(e.g., to increase shelf-life). Moreover, conjugation of a drug to acarrier protein can be difficult to control, resulting in aheterogeneous mixture of conjugates that differ in the number of drugmolecules attached. This can make controlling the amount administered toa patient difficult.

There is a need for methods and compositions that provide drugconjugates.

SUMMARY

The disclosure provides aldehyde-tagged protein carriers that can becovalently and site-specifically bound to drug to provide adrug-containing scaffold. The disclosure also provides methods ofproduction of such drug-containing scaffolds and intermediates, as wellas methods of use.

Accordingly, the present disclosure provides carrier protein-drugconjugates composed of a carrier protein and a covalently bound drug,wherein the carrier protein comprises a modified sulfatase motif of theformula:

X₁(FGly′)X₂Z₂X₃Z₃

where FGly′ is of the formula:

wherein J¹ is the covalently bound drug;

each L¹ is a divalent moiety independently selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,alkynylene, arylene, substituted arylene, cycloalkylene, substitutedcycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene,substituted heterocyclene, acyl, amido, acyloxy, urethanylene,thioester, sulfonyl, sulfonamide, sulfonyl ester, —O—, —S—, —NH—, andsubstituted amine;

n is a number selected from zero to 40;

Z₂ is a proline or alanine residue;

X₁ is present or absent and, when present, is any amino acid, with theproviso that when the sulfatase motif is at an N-terminus of thepolypeptide, X₁ is present;

X₂ and X₃ are each independently any amino acid; and

Z₃ is a basic amino acid, and

wherein the carrier protein presents the covalently bound drug on asolvent-accessible surface when in a folded state.

In some embodiments, the carrier protein-drug conjugate contains two ormore modified sulfatase motifs, and can contain three or more modifiedsulfatase motifs.

In further embodiments the modified sulfatase motifs of the carrierprotein-drug conjugate are positioned in the carrier protein-drugconjugate at at least one of an N-terminus of the carrier protein, aC-terminus of the carrier protein, and a solvent-accessible loop of thecarrier protein.

The modified sulfatase motifs of the carrier protein-drug conjugate canbe provided as a concatamer composed of modified sulfatase motifsseparated by a flexible linker.

In one exemplar, the carrier protein of the carrier protein-drugconjugate is albumin. The covalently bound drug can be a peptide drug,such as glucagon-like peptide 1 (GLP-1) or a biologically active variantthereof, or calcitonin or a biologically active variant thereof. Thecovalently bound drug of the carrier protein-drug conjugate can be asmall molecule drug (e.g., doxorubicin).

Exemplary carrier protein-drug conjugates include those where Z₃ isarginine (R). In exemplary embodiments, X₁, when present, X₂, and X₃ areeach independently an aliphatic amino acid, a sulfur-containing aminoacid, or a polar, uncharged amino acid. In specific examples, X₁, whenpresent, is L, M, V, S or T. In specific examples, X₂ and X₃ are eachindependently S, T, A, V, G, or C.

The disclosure also provides aldehyde-tagged carrier proteins having anamino acid sequence of:

X₁Z₁X₂Z₂X₃Z₃

where

Z₁ is a cysteine, a serine, or a 2-formylglycine residue;

Z₂ is a proline or alanine residue;

X₁ is present or absent and, when present, is any amino acid, with theproviso that when the heterologous sulfatase motif is at an N-terminusof the aldehyde tagged polypeptide, X₁ is present; and

X₂ and X₃ are each independently any amino acid; and

Z₃ is a basic amino acid;

wherein the carrier protein presents the covalently bound drug on asolvent-accessible surface when in a folded state.

In some examples the aldehyde-tagged carrier protein contains two ormore modified sulfatase motifs, and can contain three or more modifiedsulfatase motifs. In some examples, the modified sulfatase motifs arepositioned in the aldehyde-tagged carrier protein at at least one of anN-terminus of the carrier protein, a C-terminus of the carrier protein,and a solvent-accessible loop of the carrier protein. In one example,the carrier protein is albumin

Exemplary aldehyde-tagged carrier proteins include those in which Z₃ isarginine (R). Exemplary aldehyde-tagged carrier proteins include thosein which X₁, when present, X₂, and X₃ are each independently analiphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid. In specific examples of aldehyde-tagged carrierprotein, X₁, when present, is L, M, V, S or T. In specific examples ofaldehyde-tagged carrier protein, X₂ and X₃ are each independently S, T,A, V, G, or C.

The disclosure also provides libraries of modified carrier proteinscontaining a population of aldehyde-tagged carrier proteins according tothe present disclosure, or nucleic acid constructs encoding thealdehyde-tagged carrier proteins, where Z₁ is a cysteine or serineresidue, wherein the population comprises members having differentlyaldehyde-tagged carrier proteins. In some examples, the population ofthe library includes aldehyde-tagged carrier proteins having two or morealdehyde tags. In some examples, the wherein the population of thelibrary includes aldehyde-tagged carrier proteins having at least onealdehyde tag at one or more of the N-terminus, the C-terminus, or aninterior loop and the carrier protein. In some embodiments, the libraryis provided as a population of recombinant cells genetically modified toexpress the nucleic acid constructs.

The disclosure also provides methods of producing a carrier protein-drugconjugate by combining in a reaction mixture an aldehyde-tagged carrierprotein having a 2-formyl-glycine residue (FGly′ at Z₁) and a drug forconjugation to the carrier protein, wherein the drug has an aminooxy orhydrazide reactive group. The drug is provided in the reaction mixturein an amount sufficient to provide for a desired ratio of drug tocarrier protein, said combining being under conditions suitable topromote reaction between an aldehyde of the carrier protein and reactivegroup of the drug to generate a carrier protein-drug conjugate. Thecarrier protein-drug conjugate is then isolated from the reactionmixture. In specific embodiments, the aldehyde-tagged carrier protein isfolded prior to said combining.

The disclosure also provides formulations containing a carrierprotein-drug conjugate of the present disclosure and a pharmaceuticallyacceptable excipient.

The disclosure also provides methods of treating a subject having or atrisk of having condition amenable to treatment with glucagon-likepeptide 1 (GLP-1) by administering to a subject in of treatment acarrier protein-drug conjugate of the present disclosure in which thecovalently bound drug is glucagon-like peptide 1 (GLP-1) or abiologically active variant thereof, where administration is effectiveto treat the condition in the subject. The disclosure also providesmethods of treating a subject having or at risk of having conditionamenable to treatment with calcitonin by administering to a subject inof treatment a carrier protein-drug conjugate of the present disclosurein which the covalently bound drug is calcitonin or a biologicallyactive variant thereof, where administration is effective to treat thecondition in the subject.

The disclosure also provides recombinant nucleic acids having nucleicacid encoding an aldehyde-tagged carrier protein of the presentdisclosure in which Z₁ is a cysteine residue or a serine residue.

Other features are provided below, and will be readily apparent to theordinarily skilled artisan upon reading the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

It is emphasized that, according to common practice, the variousfeatures of the drawings may not be to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures:

FIG. 1A is a schematic illustrating production of an ald-tagged carrierprotein containing a 2-formylglycine (FGly), which is reacted with anaminooxy-containing peptide to form a drug-conjugate of the presentdisclosure. The aldehyde tag is exemplified in FIG. 1A by LCTPSR (SEQ IDNO:1).

FIG. 1B is a schematic illustrating a library of ald-tagged carrierproteins, exemplified by an ald-tagged human serum albumin (HSA) (toppanel), and ald-tagged HSA-drug conjugates (bottom panel) conjugated toa drug. By changing the location of the aldehyde tag, the spatialdisplay of the peptide relative to the surface of the rHSA can bealtered.

FIG. 2 is schematic illustrating an exemplary synthesis of peptides tocontain a reactive partner for reaction with an aldehyde group of anald-tagged carrier protein.

FIG. 3 is a schematic providing an amino acid sequence (SEQ ID NO:70)and nucleic acid sequence (SEQ ID NO:71) of human serum albumin (HSA).

FIG. 4 is a schematic providing amino acid sequences of exemplaryald-tagged HSA proteins (SEQ ID NO:72-76). The prepro leader sequence isindicated by a single underline. The sulfatase motif is indicated by adouble underline.

FIGS. 5-9 are schematics providing the nucleic acid sequences (SEQ IDNO:77-81) of the exemplary ald-tagged HSA proteins of FIG. 4.

FIG. 10 is a schematic illustrating (top panel) the crystal structure ofa recombinant HSA and (bottom panel) a carrier protein-drug conjugate ofan ald-tagged recombinant HSA and GLP-1, with the GLP-1 peptide shown inthe same scale as the HSA carrier protein. In each panel, the N-terminalend of the protein is on the right side of the schematic; the C-terminalend of the protein is on the left side of the schematic.

FIG. 11 provides amino acid sequences (SEQ ID NO:82-89) of exemplaryald-tagged Fc proteins. The sulfatase motif is indicated bydouble-underlined text.

FIG. 12 is a schematic of an exemplary ald-tagged carrier proteinmodified by conjugation to a small molecule drug. The N-terminal end ofthe protein is on the right side of the schematic; the C-terminal end ofthe protein is on the left side of the schematic. The aldehyde tag isexemplified in FIG. 12 by LCTPSR (SEQ ID NO:1).

FIG. 13 is a picture of a protein gel illustrating HSA, purified usingNi/NTA, purified using Ni/NTA, containing an aldehyde tag at the Cterminus conjugated with a fluorophore. The negative control, CtoAconstruct, does not get converted to a formylglycine and is subsequentlynot conjugated when reacted with the fluorophore.

FIG. 14 is a picture of a gel showing Ald tag-HSA was expressed in andsecreted from the yeast Pichia pastoris. A Pichia strain expressing noHSA (none), wild-type HSA (WT HSA), or ald tag-HSA (clones #7, 11, 24,25) was grown in methanol-containing medium to induce expression of HSA.After 6 days, the media was collected, cleared of cells, run on anSDS-PAGE gel, and stained with Coomassie Blue. proteing gel is a Aldtag-HSA was expressed in and secreted from the yeast Pichia pastoris. APichia strain expressing no HSA (none), wild-type HSA (WT HSA), or aldtag-HSA (clones #7, 11, 24, 25) was grown in methanol-containing mediumto induce expression of HSA. After 6 days, the media was collected,cleared of cells, run on an SDS-PAGE gel, and stained with CoomassieBlue.

FIG. 15 is a picture of a gel showing aldehyde-tagged-HSA was expressedand secreted from CHO cells. After 72 h, the media was collected,cleared of cells, and purified on Ni-NTA resin. Flow-through (FT), wash(W) and elution (E) fractions were collected, run on an SDS-PAGE gel andstained with Coomassie Blue.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “analdehyde tag” includes a plurality of such tags and reference to “thepolypeptide” includes reference to one or more polypeptides andequivalents thereof known to those skilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude anyelement which may be optional. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compoundsthat are stable compounds (i.e., compounds that can be made, isolated,characterized, and tested for biological activity). In addition, allsub-combinations of the various embodiments and elements thereof (e.g.,elements of the chemical groups listed in the embodiments describingsuch variables) are also specifically embraced by the present inventionand are disclosed herein just as if each and every such sub-combinationwas individually and explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymeric form of amino acids ofany length. Unless specifically indicated otherwise, “polypeptide”,“peptide” and “protein” can include genetically coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, proteins which contain at least oneN-terminal methionine residue (e.g., to facilitate production in arecombinant bacterial host cell); immunologically tagged proteins; andthe like.

“Native amino acid sequence” or “parent amino acid sequence” are usedinterchangeably herein in the context of a carrier protein to refer tothe amino acid sequence of the carrier protein prior to modification toinclude a heterologous aldehyde tag.

By “aldehyde tag” or “ald-tag” is meant an amino acid sequence thatcontains an amino acid sequence derived from a sulfatase motif which iscapable of being converted, or which has been converted, by action of aformylglycine generating enzyme (FGE) to contain a 2-formylglycineresidue (referred to herein as “FGly”). Although this is technicallyincorrect, the FGly residue generated by an FGE is often referred to inthe literature as a “formylglycine”. Stated differently, the term“aldehyde tag” is used herein to refer to an amino acid sequencecomprising an “unconverted” sulfatase motif (i.e., a sulfatase motif inwhich the cysteine or serine residues has not been converted to FGly byan FGE, but is capable of being converted) as well as to an amino acidsequence comprising a “converted” sulfatase motif (i.e., a sulfatasemotif in which the cysteine or serine residues has been converted toFGly by action of an FGE).

By “conversion” as used in the context of action of a formylglycinegenerating enzyme (FGE) on a sulfatase motif refers to biochemicalmodification of a cysteine or serine residue in a sulfatase motif to aformylglycine (FGly) residue (e.g., Cys to FGly, or Ser to FGly).

“Modification” encompasses addition, removal, or alteration of a moiety.As used in the context of a polypeptide having a converted sulfatasemotif, “modification” is meant to refer to chemical or biochemicalmodification of an FGly residue of an aldehyde tag of a polypeptidethrough reaction of the FGly aldehyde moiety with a reactive partner. Asdiscussed above, the term “conversion” refers to a type of biochemicalmodification of an FGly residue of an aldehyde tag mediated by an FGE.An aldehyde tag that is modified by reaction of an FGly with a reactivepartner as described herein is sometimes referred to as a “modified aldtag” or an aldehyde tag containing “FGly′”.

By “genetically-encodable” as used in reference to an amino acidsequence of polypeptide, peptide or protein means that the amino acidsequence is composed of amino acid residues that are capable ofproduction by transcription and translation of a nucleic acid encodingthe amino acid sequence, where transcription and/or translation mayoccur in a cell or in a cell-free in vitro transcription/translationsystem.

The term “control sequences” refers to DNA sequences to facilitateexpression of an operably linked coding sequence in a particularexpression system, e.g. mammalian cell, bacterial cell, cell-freesynthesis, etc. The control sequences that are suitable for prokaryotesystems, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cell systems mayutilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate the initiation of translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. Linking is accomplished by ligation or throughamplification reactions. Synthetic oligonucleotide adaptors or linkersmay be used for linking sequences in accordance with conventionalpractice.

The term “expression cassette” as used herein refers to a segment ofnucleic acid, usually DNA, that can be inserted into a nucleic acid(e.g., by use of restriction sites compatible with ligation into aconstruct of interest or by homologous recombination into a construct ofinterest or into a host cell genome). In general, the nucleic acidsegment comprises a polynucleotide that encodes a polypeptide ofinterest (e.g., an aldehyde tagged-carrier protein), and the cassetteand restriction sites are designed to facilitate insertion of thecassette in the proper reading frame for transcription and translation.Expression cassettes can also comprise elements that facilitateexpression of a polynucleotide encoding a polypeptide of interest in ahost cell. These elements may include, but are not limited to: apromoter, a minimal promoter, an enhancer, a response element, aterminator sequence, a polyadenylation sequence, and the like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,usually 75% free, and most usually 90% free from other components withwhich it is naturally associated.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

By “heterologous” is meant that a first entity and second entity areprovided in an association that is not normally found in nature. Forexample, a protein containing a “heterologous” sulfatase motif or“heterologous” ald-tag is a protein that does not normally contain asulfatase motif at that position within its amino acid sequence (e.g.,proteins which have a single, native sulfatase motif can contain asecond sulfatase motif that is “heterologous”; further proteins whichcontain a sulfatase motif can be modified so as to reposition thesulfatase motif, rendering the re-positioned sulfatase motif“heterologous” to the protein). In some embodiments, a heterologoussulfatase motif is present in a polypeptide which contains no nativesulfatase motif.

By “reactive partner” is meant a molecule or molecular moiety thatspecifically reacts with another reactive partner to produce a reactionproduct. Exemplary reactive partners include an cysteine or serine ofsulfatase motif and a formylglycine generating enzyme (FGE), which reactto form a reaction product of a converted aldehyde tag containing a FGlyin lieu of cysteine or serine in the motif. Other exemplary reactivepartners include an aldehyde of a formylglycine (FGly) residue of aconverted aldehyde tag and a reactive partner reagent comprising amoiety of interest, which react to form a reaction product of a modifiedaldehyde tagged polypeptide having the moiety of interest conjugated tothe aldehyde tagged polypeptide at the FGly residue.

“N-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free amine group, which amine group in non-N-terminus aminoacid residues normally forms part of the covalent backbone of thepolypeptide.

“C-terminus” refers to the terminal amino acid residue of a polypeptidehaving a free carboxyl group, which carboxyl group in non-C-terminusamino acid residues normally forms part of the covalent backbone of thepolypeptide.

By “N-terminal” is meant the region of a polypeptide that is closer tothe N-terminus than to the C-terminus.

By “C-terminal” is meant the region of a polypeptide that is closer tothe C-terminus than to the N-terminus.

By “internal site” as used in referenced to a polypeptide or an aminoacid sequence of a polypeptide means a region of the polypeptide that isnot at the N-terminus or at the C-terminus, and includes both N-terminaland C-terminal regions of the polypeptide.

INTRODUCTION

The present disclosure provides aldehyde-tagged protein carriers thatcan be covalently and site-specifically bound to drug to provide adrug-containing scaffold, as well as methods of production of suchdrug-containing scaffolds and intermediates, as well as methods of use.Aldehyde-tagged carrier proteins may also be referred to herein as“ald-tagged carrier proteins”, “ald-tagged protein scaffolds” or“ald-tagged scaffolds”. Such Ald-tagged scaffolds can besite-specifically decorated with a covalently bound molecule ofinterest, such as a drug (e.g., a peptide, a small molecule drug, andthe like). Such drug-scaffold conjugates can provide for enhanced serumhalf-life of the drug.

The compositions and methods of the present disclosure exploit anaturally-occurring, genetically-encodable sulfatase motif for use as atag, referred to herein as an “aldehyde tag” or “ald tag”, to directsite-specific modification of the carrier protein. The sulfatase motifof the aldehyde tag, which is based on a motif found in active sites ofsulfatases, contains a serine or cysteine residue that is capable ofbeing converted (oxidized) to a 2-formylglycine (FGly) residue by actionof a formylglycine generating enzyme (FGE) either in vivo (e.g., at thetime of translation of an ald tag-containing protein in a cell) or invitro (e.g., by contacting an ald tag-containing protein with an FGE ina cell-free system). The aldehyde moiety of the resulting FGly residuecan be used as a “chemical handle” to facilitate site-specific chemicalmodification of the protein, and thus site-specific attachment of a drugof interest. For example, a peptide modified to contain anα-nucleophile-containing moiety (e.g., an aminooxy or hydrazide moiety)can be reacted with the FGly-containing carrier protein to yield aconjugate in which the carrier protein and peptide are linked by ahydrazone or oxime bond, respectively. The reactivity of the aldehydethus allows for bioorthongonal and chemoselective modification of thecarrier protein, and thus provides a site-specific means for chemicalmodification that in turn can be exploited to provide for site-specificattachment of a moiety of interest in the final conjugate.

For illustrative purposes, a schematic of production of an ald-taggedcarrier protein is provided in FIG. 1A. In this example, a constructencoding a carrier protein having an ald tag (exemplified by LCTPSR (SEQID NO:1)) is expressed in a host cell (exemplified by the yeast S.cerevisae) which is genetically modified to contain an FGE of M.tuberculosis. Expressing the recombinant protein in yeast not modifiedwith FGE is produced without the cysteine being converted to FGly. Thecarrier protein can be purified and added to recombinant, purified FGEgenerating the aldehyde tag on the carrier protein. The resultingcarrier protein contains an FGly having an aldehyde moiety (arrow). Theald-tagged carrier protein is then reacted with a peptide having areactive aminoooxy moiety. The reaction product is a drug-carrierprotein conjugate having the peptide bound to the carrier proteinthrough an oxime bond. FIG. 1B illustrates how the ald-tag can be placedat various positions on the carrier protein, thus providing fordrug-carrier protein conjugates having bound drug (exemplified by apeptide drug) at different positions on the carrier protein.

Exemplary methods and compositions for practice of the invention willnow be described in more detail.

Aldehyde Tags

In general, an aldehyde tag (“ald tag”) can be based on any amino acidsequence derived from a sulfatase motif (also referred to as a“sulfatase domain”), which is capable of being converted by action of aformylglycine generating enzyme (FGE) to contain a formylglycine (FGly).Action of FGE is directed in a sequence-specific manner in that the FGEacts at a sulfatase motif, but this sulfatase motif can be positionedwithin any region of carrier protein. Thus, FGE-mediated conversion of asulfatase motif is site-specific (i.e., in that FGE acts at the aminoacid sequence of a sulfatase motif) but the ability of FGE to act uponthe sulfatase motif is sequence context-independent (i.e., the abilityof the FGE to convert a cysteine/serine of a sulfatase motif isindependent of the sequence context in which the sulfatase motif ispresented in the carrier protein).

Exemplary Aldehyde Tags

A minimal sulfatase motif of an aldehyde tag is usually about 5 or 6amino acid residues in length, usually no more than 6 amino acidresidues in length. In general, it is normally desirable to minimize theextent of modification of the native amino acid sequence of the carrierprotein, so as to minimize the number of amino acid residues that areinserted, deleted, substituted (replaced), or added (e.g., to the N- orC-terminus). Minimizing the extent of amino acid sequence modificationof the carrier protein is usually desirable so as to minimize the impactsuch modifications may have upon carrier protein structure and/orimmunogenicity. Thus, aldehyde tags of particular interest include thosethat require modification (insertion, addition, deletion,substitution/replacement) of less than 16, 15, 14, 13, 12, 11, 10, 9, 8,or 7 amino acid residues of the amino acid sequence of the carrierprotein (e.g., the carrier polypeptide).

It should be noted that while aldehyde tags of particular interest arethose based on a minimal sulfatase motif, it will be readily appreciatedthat longer aldehyde tags are both contemplated and encompassed by thepresent disclosure and can find use in the compositions and methods ofthe invention. Aldehyde tags can thus comprise a minimal sulfatase motifof 5 or 6 residues, or can be longer and comprise a minimal sulfatasemotif which can be flanked at the N- and/or C-terminal sides of themotif by additional amino acid residues. Aldehyde tags of, for example,5 or 6 amino acid residues are contemplated, as well as longer aminoacid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acid residues.

In general, sulfatase motifs useful in aldehyde tags as described hereinare of the formula:

X₁Z₁X₂Z₂X₃Z₃  (I)

or, in an exemplary embodiment

X₁Z₁X₂Z₂X₃R  (Ia)

where

Z₁ is cysteine or serine (which can also be represented by (C/S));

Z₂ is either a proline or alanine residue (which can also be representedby (P/A));

Z₃ is a basic amino acid, and may be arginine (R), lysine (K) orhistidine (H), usually lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),usually A, G, L, V, or I (in Formula (Ia) Z₃ is arginine (R));

X₁ is present or absent and, when present, can be any amino acid, thoughusually an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than a aromatic amino acid ora charged amino acid), usually L, M, V, S or T, more usually L, M, S orV, with the proviso that when the sulfatase motif is at the N-terminusof the carrier protein, X₁ is present; and

X₂ and X₃ independently can be any amino acid, though usually analiphatic amino acid, a polar, uncharged amino acid, or a sulfurcontaining amino acid (i.e., other than a aromatic amino acid or acharged amino acid), usually S, T, A, V, G or C, more usually S, T, A, Vor G.

It should be noted that, following action of an FGE on the sulfatasemotif, Z₁ is oxidized to generate a 2-formylglycine (FGly) residue.Furthermore, following both FGE-mediated conversion and reaction with areactive partner of a drug of interest, FGly position at Z₁ in theformula above is covalently bound to the drug (e.g., a peptide drug,etc). The reactive partner generally is an α-nucleophile, such as anaminooxy or hydrazide group, and provides for linkage of the carrierprotein to the drug through an oxime or hydrazone linkage. Thus thecarrier protein and drug are not linked through an amide bond, as may befound in other drug conjugates based on recombinant fusion proteintechnology.

Where the aldehyde tag is present at a location other than theN-terminus of the carrier protein, X₁ of the formula above can beprovided by an amino acid residue of the native amino acid sequence ofthe carrier protein. Therefore, in some embodiments, and when present ata location other than the N-terminus of a carrier protein, sulfatasemotifs are of the formula:

(C/S)X₁(P/A)X₂Z₃  (II)

or, in an exemplary embodiment

(C/S)X₁(P/A)X₂R  (IIa)

where X₁ and X₂ independently can be any amino acid, though usually analiphatic amino acid, a polar, uncharged amino acid, or asulfur-containing amino acid (i.e., other than an aromatic amino acid ora charged amino acid), usually S, T, A, V, or C, more usually S, T, A,or V. Z₃ in Formula II is defined as above.

As noted above, the sulfatase motif can contain additional residues atone or both of the N- and C-terminus of the sequence, e.g., such thatthe aldehyde tag includes both a sulfatase motif and an “auxiliarymotif”. In one embodiment, the sulfatase motif includes an auxiliarymotif at the C-terminus (i.e., following the arginine residue in theformula above) 1, 2, 3, 4, 5, 6, or all 7 of the contiguous residues ofan amino acid sequence of AALLTGR (SEQ ID NO:46), SQLLTGR (SEQ IDNO:47), AAFMTGR (SEQ ID NO:48), AAFLTGR (SEQ ID NO:49), SAFLTGR (SEQ IDNO:50), ASILTGK (SEQ ID NO:51), VSFLTGR (SEQ ID NO:52), ASLLTGL (SEQ IDNO:53), ASILITG (SEQ ID NO:54), VSFLTGR (SEQ ID NO:55), SAIMTGR (SEQ IDNO:56), SAIVTGR (SEQ ID NO:57), TNLWRG (SEQ ID NO:58), TNLWRGQ (SEQ IDNO:59), TNLCAAS (SEQ ID NO:60), VSLWTGK (SEQ ID NO:61), SMLLTG (SEQ IDNO:62), SMLLTGN (SEQ ID NO:63), SMLLTGT (SEQ ID NO:64), ASFMAGQ (SEQ IDNO:65), or ASLLTGL (SEQ ID NO:66), (see, e.g., Dierks et al. (1999) EMBOJ 18(8): 2084-2091), or of GSLFTGR (SEQ ID NO:67). Additional C-terminalamino acid residues are not required for FGE-mediated conversion of thesulfatase motif of the aldehyde tag, and thus are only optional and maybe specifically excluded from the aldehyde tags described herein. Insome embodiments the aldehyde tag does not contain an amino acidsequence CGPSR(M/A)S (SEQ ID NO:68) or CGPSR(M/A) (SEQ ID NO:69), whichmay be present as a native amino acid sequence in phosphonate monoesterhydrolases.

The sulfatase motif of the aldehyde tag is generally selected so as tobe capable of conversion by a selected FGE, e.g., an FGE present in ahost cell in which the aldehyde tagged polypeptide is expressed or anFGE which is to be contacted with the aldehyde tagged polypeptide in acell-free in vitro method.

Selection of aldehyde tags and an FGE that provide for suitable reactivepartners to provide for generation of an FGly in the aldehyde taggedcarrier protein can be readily accomplished in light of informationavailable in the art. In general, sulfatase motifs susceptible toconversion by a eukaryotic FGE contain a cysteine and a proline (i.e., acysteine and proline at Z₁ and Z₂, respectively, in Formula I above(e.g., X₁CX₂PX₃R); CX₁PX₂R in Formula II above) and are modified by the“SUMF1-type” FGE (Cosma et al. Cell 2003, 113, (4), 445-56; Dierks etal. Cell 2003, 113, (4), 435-44). Sulfatase motifs susceptible toconversion by a prokaryotic FGE contain either a cysteine or a serine,and a proline in the sulfatase motif (i.e., a cysteine or serine at Z₁,and a proline at Z₂, respectively, in Formula I above (e.g.,X₁(C/S)X₂PX₃R); (C/S)X₁PX₂R in Formula II above) are modified either bythe “SUMF1-type” FGE or the “AtsB-type” FGE, respectively (Szameit etal. J Biol Chem 1999, 274, (22), 15375-81). Other sulfatase motifssusceptible to conversion by a prokaryotic FGE contain either a cysteineor a serine, and either a proline or an alanine in the sulfatase motif(i.e., a cysteine or serine at Z₁, and a proline or alanine at Z₂,respectively, e.g, SX₁AX₂R; X₁CX₂PX₃Z₃; X₁SX₂PX₂Z₃; X₁CX₂AX₃Z₃;X₁SX₂AX₃Z₃; CX₁PX₂Z₃; SX₁PX₂Z₃; CX₁AX₂Z₃; SX₁AX₂Z₃ (in Formula I above);CX₁PX₂Z₃ (in Formula II above); X₁CX₂PX₃R; X₁SX₂PX₂R; X₁CX₂AX₃R;X₁SX₂AX₃R (in Formula Ia above); CX₁PX₂R; SX₁PX₂R; CX₁AX₂R; SX₁AX₂R (inFormula IIa above), and are susceptible to modification by, for example,can be modified by an FGE of a Firmicutes (e.g., Clostridiumperfringens) (see Berteau et al. J. Biol. Chem. 2006; 281:22464-22470).

Therefore, for example, where the FGE is a eukaryotic FGE (e.g., amammalian FGE, including a human FGE), the sulfatase motif is usually ofthe formula:

X₁CX₂PX₃Z₃  (III)

or, in an exemplary embodiment

X₁CX₂PX₃R  (IIIa)

where

X₁ may be present or absent and, when present, can be any amino acid,though usually an aliphatic amino acid, a sulfur-containing amino acid,or a polar, uncharged amino acid, (i.e., other than a aromatic aminoacid or a charged amino acid), usually L, M, S or V, with the provisothat when the sulfatase motif is at the N-terminus of the carrierprotein, X₁ is present;

X₂ and X₃ independently can be any amino acid, though usually analiphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid, (i.e., other than a aromatic amino acid or acharged amino acid), usually S, T, A, V, G, or C, more usually S, T, A,V or G; and

Z₃ is a basic amino acid (which may be other than arginine (R), and maybe lysine (K) or histidine (H), usually lysine), or an aliphatic aminoacid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I),or proline (P), usually A, G, L, V, or I, where Z₃ is arginine (R) inFormula IIIa.

Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO:1),MCTPSR (SEQ ID NO:2), VCTPSR(SEQ ID NO:3), LCSPSR (SEQ ID NO:4), LCAPSR(SEQ ID NO:5) LCVPSR (SEQ ID NO:6), LCGPSR(SEQ ID NO:7), ICTPAR(SEQ IDNO:8), LCTPSK(SEQ ID NO:9), MCTPSK (SEQ ID NO:10), VCTPSK (SEQ IDNO:11), LCSPSK (SEQ ID NO:12), LCAPSK (SEQ ID NO:13), LCVPSK(SEQ IDNO:14), LCGPSK(SEQ ID NO:15), LCTPSA (SEQ ID NO:16), ICTPAA (SEQ IDNO:17), MCTPSA (SEQ ID NO:18), VCTPSA (SEQ ID NO:19), LCSPSA (SEQ IDNO:20), LCAPSA (SEQ ID NO:21), LCVPSA (SEQ ID NO:22), and LCGPSA (SEQ IDNO:23). Other specific sulfatase motifs are readily apparent from thedisclosure provided herein.

As described in more detail below, a converted aldehyde taggedpolypeptide is reacted with a reactive partner of a moiety of interestto provide for conjugation between the moiety of interest to the FGlyresidue of the converted aldehyde tagged polypeptide, and production ofa modified polypeptide (e.g., a conjugate of the ald-tagged carrierprotein and a peptide drug). Modified polypeptides having a modifiedaldehyde tag are generally described by comprising a modified sulfatasemotif of the formula:

X₁(FGly′)X₂Z₂X₃Z₃  (I′)

or, in an exemplary embodiment

X₁(FGly′)X₂Z₂X₃R  (Ia′)

where

FGly′ is a formylglycine residue having a covalently attached moiety(e.g., a peptide drug);

Z₂ is either a proline or alanine residue (which can also be representedby (P/A));

Z₃ in Formula I′ is a basic amino acid, and may be arginine (R) (as inFormula Ia′), lysine (K) or histidine (H), usually lysine), or analiphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V),isoleucine (I), or proline (P), usually A, G, L, V, or I;

X₁ may be present or absent and, when present, can be any amino acid,though usually an aliphatic amino acid, a sulfur-containing amino acid,or a polar, uncharged amino acid, (i.e., other than a aromatic aminoacid or a charged amino acid), usually L, M, V, S or T, more usually L,M or V, with the proviso that when the sulfatase motif is at theN-terminus of the carrier protein, X₁ is present; and

X₂ and X₃ independently can be any amino acid, though usually analiphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid, (i.e., other than a aromatic amino acid or acharged amino acid), usually S, T, A, V, G or C, more usually S, T, A, Vor G.

Specific examples of converted sulfatase motifs include L(FGly)TPSR (SEQID NO:24), M(FGly)TPSR (SEQ ID NO:25), V(FGly)TPSR (SEQ ID NO:26),L(FGly)SPSR (SEQ ID NO:27), L(FGly)APSR (SEQ ID NO:28), L(FGly)VPSR (SEQID NO:29), L(FGly)GPSR (SEQ ID NO:30), I(FGly)TPAR (SEQ ID NO:31),L(FGly)TPSK (SEQ ID NO:32), M(FGly)TPSK (SEQ ID NO:33), V(FGly)TPSK (SEQID NO:34), L(FGly)SPSK (SEQ ID NO:35), L(FGly)APSK (SEQ ID NO:36),L(FGly)VPSK (SEQ ID NO:37), L(FGly)GPSK (SEQ ID NO:38), L(FGly)TPSA (SEQID NO:39), M(FGly)TPSA (SEQ ID NO:40), V(FGly)TPSA (SEQ ID NO:41),L(FGly)SPSA (SEQ ID NO:42), L(FGly)APSA (SEQ ID NO:43), L(FGly)VPSA (SEQID NO:44), and L(FGly)GPSA (SEQ ID NO:45). It will be appreciated thatexemplary carrier proteins that are covalently bound to drug throughreaction with the aldehyde of the FGly residue include those having theamino acid sequences described above, but the modified FGly (representedabove by FGly′) in lieu of the unmodified FGly.

Carrier Proteins

In general a “carrier protein” is a protein that is biologically inert,is susceptible to modification by use of the ald tag technology asdisclosed herein, and which can provide for solvent-accessiblepresentation of drug conjugated to the carrier protein through amodified ald-tag positioned in the carrier protein (e.g., through anoxime or hydrazone bond within the converted sulfatase motif of the aldtagged carrier protein) in a physiological environment. “Biologicallyinert” is meant to indicate the carrier protein exhibits clinicallyinsignificant or no detectable biological activity when administered tothe appropriate subject, particularly when administered to a humansubject. Thus, carrier proteins are biologically inert in that they, forexample, are of low immunogenicity, do not exhibit significant ordetectable targeting properties (e.g., do not exhibit significant ordetectable activity in binding to a specific receptor), and exhibitlittle or no detectable biological activity that may interfere withactivity of a drug to be conjugated to the ald-tagged carrier protein.By “low immunogenicity” is meant that the carrier protein elicits littleor no detectable immune response upon administration to a subject,especially a mammalian subject, more especially a human subject. Carrierproteins can be provided in monomeric or multimeric (e.g., dimeric)forms.

Carrier proteins having a three-dimensional structure when folded thatprovides for multiple different solvent-accessible sites that areamenable to ald-tag modification (and thus conjugation to a drug) are ofparticular interest. In general, carrier proteins of interest are thosethat are of a size and three-dimensional folded structure so as toprovide for presentation of conjugated drug on solvent accessiblesurfaces in a manner that is sufficient spatially separated so as toprovide for activity and bioavailability of the conjugated drugmolecules are of particular interest. The carrier protein will beselected according to a variety of factors including, but not limitedto, the drug to be conjugated to the carrier.

Accordingly, any of a wide variety of polypeptides can be suitable foruse as ald-tagged carrier proteins for use in the drug-carrier proteinsconjugates of the present disclosure. Such carrier proteins can includethose having a naturally-occurring amino acid sequence, a native aminoacid sequence having an N-terminal methionine, fragments ofnaturally-occurring polypeptides, and non-naturally occurringpolypeptides and fragments thereof.

Exemplary carrier proteins include, but are not necessarily limited to,albumin and fragments thereof (e.g., human serum albumin, bovine serumalbumin, and the like), transferrin and fragments thereof (e.g. humantransferrin), and Fc fragments having reduced binding to a mammalian Fcreceptor, particularly a human Fc receptor (e.g., a modified Fc fragmentof an antibody (e.g., IgG), particularly a mammalian antibody, e.g., ahuman antibody). Exemplary modified Fc fragments having reduced Fcreceptor binding are exemplified by the Fc fragments of Herceptin(trastuzumab) and Rituxan (Rituximab), which contain point mutationsthat provide for reduced Fc receptor binding (see, e.g., Clynes et alNature Medicine 2000, 6, 443-446). Alternatively or in addition, theisotype of the Fc fragment can be selected according to a desired levelof Fc receptor binding (e.g., use of an Fc fragment of an IgG4 isotypehuman heavy chain constant region rather than from IgG1 or IgG3. (see,e.g, Fridman FASEB J 1991 September; 5 (12): 2684-90) In general,carrier proteins can be at least about 4 kDa (e.g., about 50 amino acidresidues in length), usually at least about 25 kDa, and can be larger insize (e.g., transferrin has a molecular weight of 90 kDa while Fcfragments can have molecular weights of 30 kDa to 50 kDa).

Modification of Carrier Proteins to Contain an Aldehyde Tag

An aldehyde tag can be provided in a carrier protein by insertion (e.g.,so as to provide a 5 or 6 amino acid residue insertion within the nativeamino acid sequence) and/or by addition (e.g., at an N- or C-terminus ofthe carrier protein). An aldehyde tag can also be provided by completeor partial substitution of native amino acid residues of the carrierprotein with the contiguous amino acid sequence of an aldehyde tag. Forexample, a heterologous aldehyde tag of 5 (or 6) amino acid residues canbe provided in a carrier protein by replacing 1, 2, 3, 4, or 5 (or 1, 2,3, 4, 5, or 6) amino acid residues of the native amino acid sequencewith the corresponding amino acid residues of the aldehyde tag.

Modification of a carrier protein to include one or more aldehyde tagscan be accomplished using recombinant molecular genetic techniques, soas produce nucleic acid encoding the desired aldehyde tagged carrierprotein. Such methods are well known in the art, and include cloningmethods, site-specific mutation methods, and the like (see, e.g.,Sambrook et al., In “Molecular Cloning: A Laboratory Manual” (ColdSpring Harbor Laboratory Press 1989); “Current Protocols in MolecularBiology” (eds., Ausubel et al.; Greene Publishing Associates, Inc., andJohn Wiley & Sons, Inc. 1990 and supplements). Alternatively, analdehyde tag can be added using non-recombinant techniques, e.g., usingnative chemical ligation or pseudo-native chemical ligation, e.g., toadd an aldehyde tag to a C-terminus of the carrier protein (see, e.g.,U.S. Pat. No. 6,184,344; U.S. Pat. No. 6,307,018; U.S. Pat. No.6,451,543; U.S. Pat. No. 6,570,040; US 2006/0173159; US 2006/0149039).See also Rush et al. (Jan. 5, 2006) Org Lett. 8(1):131-4.

Aldehyde tags can be positioned at any suitable location within acarrier protein, with the proviso that the site of the aldehyde tag isaccessible for conversion by an FGE and subsequent modification at theFGly, or can be rendered accessible (e.g., by denaturing the protein).The carrier protein can include one or more aldehyde tags. The number ofaldehyde tags that can be present in a carrier protein will vary withthe carrier protein selected, and may include 1, 2, 3, 4, 5, or morealdehyde tags.

Carrier Proteins Containing Multiple Ald Tags

Multiple ald tags can be positioned in the ald-tagged carrier protein soas to distribute the tags over the surface of the folded carrierprotein. Where the carrier protein is modified to contain multiple aldtags, the ald tags can be spaced apart in the carrier protein by aminoacid residues native to the carrier protein. Alternatively or inaddition, the carrier protein can include ald tags spaced apart by alinker, where the linker has an amino acid sequence heterologous to thecarrier protein.

Alternatively or in addition, the ald tags can be provided in theald-tagged carrier protein as a concatameric construct of 2, 3, 4 ormore ald tags, where the expression construct thus encodes for 2, 3, 4or more sulfatase motifs in a contiguous sequence of the modifiedcarrier protein, wherein the sulfatase motifs are separated by a linker]The linkers of the concatemeric constructs may be designed so as tofacilitate presentation of drug conjugated to the ald tag in the finalcarrier protein-drug conjugate. For example, the linker can be selectedso as to provide flexibility between the ald tags, thus allowing forrotation of covalently-bound drug molecules so as to enhancepresentation of biologically active drug on the carrier protein-drugconjugate surface. Such linkers can also be used in where the ald tagsare not provided as a concatamer, e.g., where an ald tag is positionedat a C- or N-terminus of a carrier protein. Ald tags, including thoseprovided as concatamers, can be positioned at or near the C-terminus ofthe carrier protein, at or near the N-terminus of the carrier protein,and/or in one or more solvent-accessible loops of the carrier protein.

Linkers will be selected according to a variety of factors (e.g., theald tag used, the number of ald tags in the concatamer, the degree offlexibility desired), and will be variable length, such as from about 3amino acids to about 25 amino acids, including about 4 amino acids toabout 23 amino acids, about 5 amino acids to about 20 amino acids, about6 amino acids to about 18 amino acids, about 7 amino acids to about 16amino acids, about 8 amino acids to about 14 amino acids, and about 9amino acids to about 12 amino acids. Exemplary flexible linkers includeglycine polymers (G)_(n), glycine-serine polymers (including, forexample, (GS)_(n), (GSGGS)_(n) (SEQ ID NO:90) and (GGGS)_(n) (SEQ IDNO:91), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers such asthe tether for the shaker potassium channel, and a large variety ofother flexible linkers, as will be appreciated by those in the art.Glycine and glycine-serine polymers are of interest since both of theseamino acids are relatively unstructured, and therefore may be able toserve as a neutral tether between components. Glycine polymers are ofparticular interests glycine accesses significantly more phi-psi spacethan even alanine, and is much less restricted tan residues with longerside chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include, but are not limitedGly-Gly-Ser-Gly-Gly (SEQ ID NO:92), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:93),Gly-Ser-Gly-Gly-Gly (SEQ ID NO:94), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:95),Gly-Ser-Ser-Ser-Gly (SEQ ID NO:96), and the like.

Concatameric ald tag constructs containing a linker can be described bythe general formula:

T₁-L_(n)-T₂

where T₁ and T₂ are the same or different ald tags as described herein(see, e.g., formulae I, Ia, I′, Ia′, II, IIa, III, and Ma), L is alinker peptide, and n is an integer of 1 or more, and may be 2, 3, 4, 5,6, 7, 8 or more. An exemplary amino acid sequence of a concatameric aldtag containing a linker is LCTPSR GGGG LCTPSR (SEQ ID NO:97), where thecysteine (C) is modified to an FGly by action of an FGE, and can bereacted with a reactive partner-containing drug to provide forcovalently bound drug as described herein.

The aldehyde tag(s) can be positioned in the carrier protein so as totake into account its structure when folded (e.g., in a cell-freeenvironment, usually a cell-free physiological environment), e.g., so asto provide the aldehyde tag at a solvent accessible site in the foldedcarrier protein. The solvent accessible aldehyde tag can thus beaccessed in the folded, unconverted ald-tagged carrier protein so as tobe accessible to an FGE for conversion of the serine or cysteine to anFGly and/or to a reactive partner reagent for conjugation to a drug ofinterest. Where an aldehyde tag is positioned at a solvent accessiblesite, in vitro FGE-mediated conversion and modification with a moiety byreaction with a reactive partner can be performed without the need todenature the protein. Solvent accessible sites can also include carrierprotein regions that are exposed at an extracellular or intracellularcell surface when expressed in a host cell.

Accordingly, or more aldehyde tags can be provided at sitesindependently selected from, for example, a solvent accessibleN-terminus, a solvent accessible N-terminal region, a solvent accessibleC-terminus, a solvent accessible C-terminal region, and/or a loopstructure. In some embodiments, the aldehyde tag is positioned at a siteother than the C-terminus of the polypeptide. In other embodiments, thepolypeptide in which the aldehyde tag is positioned is a full-lengthpolypeptide.

In other embodiments, an aldehyde tag site is positioned at a site whichis post-translationally modified in the parent carrier protein (e.g., anaturally-occurring site). For example, an aldehyde tag can beintroduced at a site of glycosylation (e.g., N-glycosylation,O-glycosylation), phosphorylation, sulftation, ubiquitination,acylation, methylation, prenylation, hydroxylation, carboxylation, andthe like in the native carrier protein. In addition or alternatively thesite of post-translational modification can be one that has beenengineered (e.g., through recombinant techniques) and does not occurnaturally in the carrier protein.

Nucleic and amino acid sequences of polypeptides suitable for use asald-tagged carrier proteins as described herein are available in theart. For example, FIG. 3 provides the amino acid sequence and encodingnucleic acid sequence for human serum albumin (HSA). Once provided theguidance of the present disclosure, the ordinarily skilled artisan canreadily generate ald-tagged HSA useful in the methods and compositionsdisclosed herein. Exemplary ald-tagged HSA amino acid and encodingnucleic acid sequences are provided in FIG. 4. Exemplary ald-tagged HSAamino acid sequences are provided in FIG. 4, with the correspondingencoding nucleic acid sequences provided in FIGS. 5-9. Thethree-dimensional structure of HSA is provided in the top panel of FIG.10.

Further exemplary ald-tagged carrier proteins include ald-tagged Fcfragment. FIG. 11 provides the amino acid sequences of exemplaryald-tagged mouse IgG1 Fc fragments having single and multiple ald tags,including exemplary ald-tagged Fc fragments containing an ald tagconcatmer with two ald tags separated by a linker.

Ald-Tagged Carrier Protein Libraries

As exemplified in the schematic of FIG. 1B, the carrier protein can bemodified to contain an ald tag at different positions to provide alibrary composed of differently ald-tagged carrier proteins, e.g.,ald-tagged carrier proteins having an ald-tag at one or more of theN-terminus, the C-terminus, an interior loop and the like. The membersof the ald-tagged carrier protein library can contain 1, 2, 3, 4, 5, ormore ald-tags. The library can be provided as a population of expressionconstructs encoding such ald-tagged carrier proteins for introductioninto host cells for expression, e.g., a host cell that expresses ancompatible FGE to provide for production of FGly-containing carrierproteins. Alternatively or in addition, the library can be provided as apopulation or recombinant host cells that are genetically modified toexpress the ald-tagged carrier protein and which, optionally, express acompatible FGE.

Such libraries can serve as a “plug and play” system for reaction of theproduced ald-tagged carrier proteins with a candidate drug having areactive partner (e.g., an aminooxy or hydrazide moiety). The reactionproductions of drug-carrier protein conjugates can then be screened fordesired characteristics (e.g., biological activity of the drug, lowimmunogenicity of the conjugate, and the like).

Formylglycine Generating Enzymes (FGEs)

A formylglycine generating enzyme (FGE) is an enzyme that oxidizescysteine or serine in a sulfatase motif to FGly. It should be noted thatin general, the literature refers to FGly-generating enzymes thatconvert a cysteine (C to FGly in a sulfatase motif as FGEs, and refersto enzymes that convert serine (S) to FGly in a sulfatase motif asAts-B-like. However, for purposes of the present disclosure “FGE” isused generically to refer to both types of FGly-generating enzymes, withthe understanding that an appropriate FGE will be selected according tothe sulfatase motif (i.e., C-containing or S-containing) present in themodified carrier protein.

In general, the FGE used to facilitate conversion of cysteine or serineto FGly in a sulfatase motif of an aldehyde tag of a carrier protein isselected according to the sulfatase motif present in the aldehyde tag.The FGE can be native to the host cell in which the aldehyde taggedcarrier protein is expressed, or the host cell can be geneticallymodified to express an appropriate FGE. Eukaryotic sulfatases generallycontain a cysteine in their sulfatase motif and are modified by the“SUMF1-type” FGE (Cosma et al. Cell 2003, 113, (4), 445-56; Dierks etal. Cell 2003, 113, (4), 435-44). Prokaryotic sulfatases generallycontain either a cysteine or a serine in their sulfatase motif and aremodified either by the “SUMF1-type” FGE or the “AtsB-type” FGE,respectively (Szameit et al. J Biol Chem 1999, 274, (22), 15375-81). AnFGE has been described in Mycobacterium tuberculosis (see, e.e.g GenBankAcc. No. NP_215226 (gi:15607852) and WO 2008/036350). FGEs have alsobeen described in deuterostomia, including vertebrates and echinodermata(see, e.g., Pepe et al. (2003) Cell 113, 445-456, Dierks et al. (2003)Cell 113, 435-444; Cosma et al. (2004) Hum. Mutat. 23, 576-581). In someembodiments it may be desired to use a sulfatase motif compatible with ahuman FGE (e.g., the SUMF1-type FGE, see, e.g., Cosma et al. Cell 113,445-56 (2003); Dierks et al. Cell 113, 435-44 (2003)), and express thealdehyde tagged protein in a human cell that expresses the FGE or in ahost cell, usually a mammalian cell, genetically modified to express ahuman FGE.

In general, an FGE for use in the methods disclosed herein can beobtained from naturally occurring sources or synthetically produced. Forexample, an appropriate FGE can be derived from biological sources whichnaturally produce an FGE or which are genetically modified to express arecombinant gene encoding an FGE. Nucleic acids encoding a number ofFGEs are known in the art and readily available (see, e.g., Preusser etal. 2005 J. Biol. Chem. 280(15):14900-10 (Epub 2005 Jan. 18); Fang etal. 2004 J Biol Chem. 79(15):14570-8 (Epub 2004 Jan. 28); Landgrebe etal. Gene. 2003 Oct. 16; 316:47-56; Dierks et al. 1998 FEBS Lett.423(1):61-5; Dierks et al. Cell. 2003 May 16; 113(4):435-44; Cosma etal. (2003 May 16) Cell 113(4):445-56; Baenziger (2003 May 16) Cell113(4):421-2 (review); Dierks et al. Cell. 2005 May 20; 121(4):541-52;Roeser et al. (2006 Jan. 3)Proc Natl Acad Sci USA 103(1):81-6; Sardielloet al. (2005 Nov. 1) Hum Mol Genet. 14(21):3203-17; WO 2004/072275;GenBank Accession No. NM_182760; and WO 2008/036350). Accordingly, thedisclosure here provides for recombinant host cells genetically modifiedto express an FGE that is compatible for use with an aldehyde tag of atagged carrier protein.

Where a cell-free method is used to convert a sulfatase motif-containingcarrier protein, an isolated FGE can be used. Any convenient proteinpurification procedures may be used to isolate an FGE, see, e.g., Guideto Protein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may prepared from a cell the produces a desired FGE,and purified using HPLC, exclusion chromatography, gel electrophoresis,affinity chromatography, and the like.

Expression Vectors and Host Cells for Production of AldehydeTagged-Carrier Polypeptides

The present disclosure provides nucleic acid encoding ald-tagged carrierpolypeptides, as well as constructs and host cells containing nucleicacid. Such nucleic acids comprise a sequence of DNA having an openreading frame that encodes an aldehyde tagged carrier protein and, inmost embodiments, is capable, under appropriate conditions, of beingexpressed. “Nucleic acid” encompasses DNA, cDNA, mRNA, and vectorscomprising such nucleic acids.

Nucleic acids contemplated herein can be provided as part of a vector(also referred to as a construct), a wide variety of which are known inthe art and need not be elaborated upon herein. Exemplary vectorsinclude, but are not limited to, plasmids; cosmids; viral vectors (e.g.,retroviral vectors); non-viral vectors; artificial chromosomes (YAC's,BAC's, etc.); mini-chromosomes; and the like. The choice of vector willdepend upon a variety of factors such as the type of cell in whichpropagation is desired and the purpose of propagation.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding a polypeptide of interest (e.g., analdehyde tagged polypeptide, an FGE, etc.), may provide for propagatingthe subject nucleic acids, or both.

Exemplary vectors that may be used include but are not limited to thosederived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 andthe M13 mp series of vectors may be used. Bacteriophage vectors mayinclude λgt10, λgt11, λgt18-23, λZAP/R and the EMBL series ofbacteriophage vectors. Cosmid vectors that may be utilized include, butare not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL,pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series ofvectors. Alternatively, recombinant virus vectors may be engineered,including but not limited to those derived from viruses such as herpesvirus, retroviruses, vaccinia virus, poxviruses, adenoviruses,adeno-associated viruses or bovine papilloma virus.

For expression of a polypeptide of interest, an expression cassette maybe employed. Thus, the present invention provides a recombinantexpression vector comprising a subject nucleic acid. The expressionvector provides a transcriptional and translational regulatory sequence,and may provide for inducible or constitutive expression, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native tothe gene encoding the polypeptide (e.g., the carrier protein or theFGE), or may be derived from exogenous sources. In general, thetranscriptional and translational regulatory sequences may include, butare not limited to, promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, and enhancer or activator sequences. In addition toconstitutive and inducible promoters, strong promoters (e.g., T7, CMV,and the like) find use in the constructs described herein, particularlywhere high expression levels are desired in an in vivo (cell-based) orin an in vitro expression system. Further exemplary promoters includemouse mammary tumor virus (MMTV) promoters, Rous sarcoma virus (RSV)promoters, adenovirus promoters, the promoter from the immediate earlygene of human CMV (Boshart et al., Cell 41:521-530, 1985), and thepromoter from the long terminal repeat (LTR) of RSV (Gorman et al.,Proc. Natl. Acad. Sci. USA 79:6777-6781, 1982). The promoter can also beprovided by, for example, a 5′UTR of a retrovirus.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host may be present to facilitate selection of cellscontaining the vector. In addition, the expression construct may includeadditional elements. For example, the expression vector may have one ortwo replication systems, thus allowing it to be maintained in organisms,for example in mammalian or insect cells for expression and in aprokaryotic host for cloning and amplification. In addition theexpression construct may contain a selectable marker gene to allow theselection of transformed host cells. Selection genes are well known inthe art and will vary with the host cell used.

Expression constructs encoding aldehyde tagged carrier proteins can alsobe generated using amplification methods (e.g., polymerase chainreaction (PCR)), where at least one amplification primer (i.e., at leastone of a forward or reverse primer) includes a nucleic acid sequenceencoding an aldehyde tag. For example, an amplification primer having analdehyde tag-encoding sequence is designed to provide for amplificationof a nucleic acid encoding a carrier protein of interest. The extensionproduct that results from polymerase-mediated synthesis from thealdehyde tag-containing forward primer produces a nucleic acidamplification product encoding a fusion protein composed of an aldehydetagged-carrier protein. The amplification product is then inserted intoan expression construct of choice to provide an aldehyde taggedpolypeptide expression construct.

Host Cells

Any of a number of suitable host cells can be used in the production ofan aldehyde tagged carrier protein. The host cell used for production ofan aldehyde tagged-carrier protein can optionally provide forFGE-mediated conversion, so that the polypeptide produced contains anFGly-containing aldehyde tag following expression and post-translationalmodification by FGE. Alternatively the host cell can provide forproduction of an unconverted aldehyde tagged carrier protein (e.g., dueto lack of expression of an FGE that facilitates conversion of thealdehyde tag).

In general, the polypeptides described herein may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. Thus, the present inventionfurther provides a host cell, e.g., a genetically modified host cellthat comprises a nucleic acid encoding an aldehyde tagged polypeptide.The host cell can further optionally comprise a recombinant FGE, whichmay be endogenous or heterologous to the host cell.

Host cells for production (including large scale production) of anunconverted or (where the host cell expresses a suitable FGE) convertedaldehyde tagged carrier protein, or for production of an FGE (e.g., foruse in a cell-free method) can be selected from any of a variety ofavailable host cells. Exemplary host cells include those of aprokaryotic or eukaryotic unicellular organism, such as bacteria (e.g.,Escherichia coli strains, Bacillus spp. (e.g., B. subtilis), and thelike) yeast or fungi (e.g., S. cerevisiae, Pichia spp., and the like),and other such host cells can be used. Exemplary host cells originallyderived from a higher organism such as insects, vertebrates,particularly mammals, (e.g. CHO, HEK, and the like), may be used as theexpression host cells.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories are provided below.

The product can be recovered by any appropriate means known in the art.Further, any convenient protein purification procedures may be employed,where suitable protein purification methodologies are described in Guideto Protein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may prepared from a cell comprising the expressionvector expressing the ald-tagged carrier protein, and purified usingHPLC, exclusion chromatography, gel electrophoresis, affinitychromatography, and the like.

Methods for Conversion and Modification of an Aldehyde Tag

Conversion of an aldehyde tag present in an aldehyde tagged carrierprotein can be accomplished by cell-based (in vivo) or cell-free methods(in vitro). Similarly, modification of a converted aldehyde tag of analdehyde tagged polypeptide can be accomplished by cell-based (in vivo)or cell-free methods (in vitro). These are described in more detailbelow.

“In Vivo” Host Cells Conversion and Modification

Conversion of an aldehyde tag of an aldehyde tagged polypeptide can beaccomplished by expression of the aldehyde tagged polypeptide in a cellthat contains a suitable FGE. In this embodiment, conversion of thecysteine or serine of the aldehyde tag is occurs during or followingtranslation in the host cell. The FGE of the host cell can be endogenousto the host cell, or the host cell can be recombinant for a suitable FGEthat is heterologous to the host cell. FGE expression can be provided byan expression system endogenous to the FGE gene (e.g., expression isprovided by a promoter and other control elements present in the nativeFGE gene of the host cell), or can be provided by from a recombinantexpression system in which the FGE coding sequence is operably linked toa heterologous promoter to provide for constitutive or inducibleexpression.

Conditions suitable for use to accomplish conjugation of a reactivepartner moiety to an aldehyde tagged polypeptide are similar to thosedescribed in Mahal et al. (1997 May 16) Science 276(5315):1125-8.

“In vitro” (Cell-Free) Conversion and Modification

In vitro (cell-free) conversion of an aldehyde tag of an aldehyde taggedcarrier protein can be accomplished by contacting an aldehyde taggedpolypeptide with an FGE under conditions suitable for conversion of acysteine or serine of a sulfatase motif of the aldehyde tag to a FGly.For example, nucleic acid encoding an aldehyde tagged polypeptide can beexpressed in an in vitro transcription/translation system in thepresence of a suitable FGE to provide for production of convertedaldehyde tagged polypeptides.

Alternatively, isolated, unconverted aldehyde tagged carrier protein canbe isolated following recombinant production in a host cell lacking asuitable FGE or by synthetic production. The isolated aldehyde taggedcarrier protein is then contacted with a suitable FGE under conditionsto provide for aldehyde tag conversion. The aldehyde tagged carrierprotein can be unfolded by methods known in the art (e.g., using heat,adjustment of pH, chaotropic agents, (e.g., urea, and the like), organicsolvents (e.g., hydrocarbons: octane, benzene, chloroform), etc.) andthe denatured protein contacted with a suitable FGE. The ald-taggedcarrier protein can then be refolded under suitable conditions.

With respect to modification of converted aldehyde tagged, modificationis normally carried out in vitro. Converted aldehyde tagged carrierprotein is isolated from a production source (e.g., recombinant hostcell production, synthetic production), and contacted with a reactivepartner-containing drug under conditions suitable to provide forconjugation of a moiety of the reactive partner to the FGly of thealdehyde tag.

Drugs for Conjugation to Ald-Tagged Carrier Proteins

Any of a number of drugs are suitable for use, or can be modified to berendered suitable for use, as a reactive partner to conjugate to an aldtagged-carrier protein. Exemplary drugs include small molecule drugs andpeptide drugs.

“Small molecule drug” as used herein refers to compound, usually anorganic compound, which exhibits a pharmaceutical activity of interestand which is generally of a molecular weight of no greater than about800 Da, and usually no greater than 2000 Da, but can encompass moleculesof up to 5 kDa and can be as large as about 10 kDa. A small inorganicmolecule refers to a molecule containing no carbon atoms, while a smallorganic molecules refers to a compound containing at least one carbonatom.

“Peptide drug” as used herein refers to amino-acid containing polymericcompounds, and is meant to encompass naturally-occurring andnon-naturally-occurring peptides, oligopeptides, cyclic peptides,polypeptides, and proteins, as well as peptide mimetics. The peptidedrugs may be obtained by chemical synthesis or be produced from agenetically encoded source (e.g., recombinant source). Peptide drugs canrange in molecular weight, and can be from 200 Da to 10 kDa or greaterin molecular weight.

Glucagon-like peptide 1 (GLP-1), calcitonin, and biologically activefragments and variants thereof are exemplary peptide drugs. By “variant”is meant a polypeptide that has an amino acid sequence that is not foundin nature, and includes polypeptides having one or more amino acidsubstitutions, insertions and/or deletions relative to anaturally-occurring parent polypeptide. “Variant” polypeptides thusencompass a polypeptide having an N- or C-terminal truncation relativeto a parent polypeptide. A “fragment” of a polypeptide is one thatshares an amino acid sequence of a naturally-occurring polypeptide, butthat is truncated at the N-terminus, C-terminus or both relative to anaturally-occurring parent polypeptide.

GLP-1 is one of several naturally occurring incretin compounds thatpossess biologic activity when released from the gut during digestion.GLP-1 naturally works on several deficient organs to lower blood sugarlevels. It is able to significantly delay the progression of Type 2diabetes, and is useful in treatment of hyperglycemis. Currently, GLP-1is less useful as a drug because it is broken down within minutes by theenzyme DPP-4, which is present throughout the human body. Coupling ofGLP1 to an ald-tagged carrier protein can provide for increased serumhalf-lifeGLP-1 and biologically active fragments and variants thereofrepresent an exemplary peptide drug of interest for conjugation to aald-tagged carrier protein of the present disclosure. Exemplaryfragments and variants of GLP-1 include, but are not necessarily limitedto, those described in Green et al. 2007 Best Pract Res Clin EndocrinolMetab 21:497-516; Brubaker et al. 2007 Trends Endocrinol Metab18:240-245; Boyle et al. 2007 J Am Osteopath Assoc 107(Suppl):S10-S16;and Drucker et al. 2006 The incretin system: glucagon-like peptide-1receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2diabetes. Lancet 368:1696-1705 32. Exemplary biologically active GLP-1variants include those having, for example, an amino acid substitutionat amino acid residues His(7), Ala(8), or Glu(9) of the native GLP-1amino acid sequence. Specific examples include (D-His7)GLP-1,(D-Ala8)GLP-1, (Gly8)GLP-1, (Ser8)GLP-1, (Aha8)GLP-1, (Thr8)GLP-1,(Aib8)GLP-1, (Abu8)GLP-1, (Va18)GLP-1, (Asp9)GLP-1, (Ala9)GLP-1,(Pro9)GLP-1, (Phe9)GLP-1, and (Lys9)GLP-1. Specific exemplarybiologically active GLP-1 variants are known as Exenatide, LY548806,CJC-1131, and Lirglutide.

Calcitonin and biologically active variants thereof represent anexemplary peptide drug of interest for conjugation to an ald-taggedcarrier protein of the present disclosure. Calcitonin is a 32 amino-acidlinear polypeptide hormone that is produced in humans primarily by theparafollicular (also known as C-cells) of the thyroid. Calcitonin hasshort absorption and elimination half-lives of 10-15 minutes and 50-80minutes, respectively and can be used therapeutically for the treatmentof hypercalcaemia or osteoporosis. Conjugation of calcintonin to anald-tagged carrier protein as disclosed herein can provide for enhancedserum half-life, and thus provide for a therapeutic that can beadministered much less frequently then the peptide alone. Exemplarybiologically active calcitonin variants include, but are not necessarilylimited to, those described in Fowler et al. Proc Natl Acad Sci USA.2005 Jul. 19; 102(29):10105-10.

The biological activity of drug conjugated to an ald-tagged carrierprotein as disclosed herein can be assayed according to methods known inthe art. Such conjugated drugs that retain at least one desiredpharmacologic activity of the corresponding parent compound are ofinterest.

Methods for Modification of Drugs to Contain Reactive Partner forReaction with 2-Formylglycine

Peptide drugs to be conjugated to an ald-tagged carrier protein aremodified to incorporate a reactive partner for reaction with an aldehydeof the FGly residue of the ald-tagged carrier protein. Since the methodsof ald-tagged polypeptide modification are compatible with conventionalchemical processes, any of a wide variety of commercially availablereagents can be used to accomplish conjugation. For example, aminooxy,hydrazide, hydrazine, or thiosemicarbazide derivatives of a number ofmoieties of interest are suitable reactive partners, and are readilyavailable or can be generated using standard chemical methods.

Where the drug is a peptide drug, the reactive moiety (e.g., aminooxy orhydrazide can be positioned at an N-terminal region, the N-terminus, aC-terminal region, the C-terminus, or at a position internal to thepeptide. FIG. 2 provides a schematic of an exemplary method forsynthesizing a peptide drug having an aminooxy group. In this example,the peptide is synthesized from a Boc-protected precursor. An aminogroup of a peptide can react with a compound comprising a carboxylicacid group and oxy-N-Boc group. As shown in FIG. 2 for example, theamino group of the peptide reacts with3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on thecompound comprising a carboxylic acid group and oxy-N-protecting groupcan include different number of carbons in the alkylene linker andsubstituents on the alkylene linker. The reaction between the aminogroup of the peptide and the compound comprising a carboxylic acid groupand oxy-N-protecting group occurs through standard peptide couplingchemistry. Examples of peptide coupling reagents that can be usedinclude, but not limited to, DCC (dicyclohexylcarbodiimide), DIC(diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP(1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC(1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuricfluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidiniumhexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl-N,N,N′,N-tetramethyluronium hexafluorophosphate),TBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), TSTU(O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),HATU(N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide), BOP-Cl(bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphoniumtetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphoniumhexafluorophosphate), DEPBT(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP(bromotris(pyrrolidino)phosphonium hexafluorophosphate). In FIG. 2, HOBtand DIC are used as peptide coupling reagents.

Deprotection to expose the amino-oxy functionality is performed on thepeptide comprising an N-protecting group. Deprotection of theN-oxysuccinimide group, for example, occurs according to standarddeprotection conditions for a cyclic amide group. Deprotectingconditions can be found in Greene and Wuts, Protective Groups in OrganicChemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al.Certain deprotection conditions include a hydrazine reagent, aminoreagent, or sodium borohydride. In FIG. 2, the deprotection of the Bocprotecting group occurs with TFA. Other reagents for deprotectioninclude, but are not limited to, hydrazine, methylhydrazine,phenylhydrazine, sodium borohydride, and methylamine. The product andintermediates can be purified by conventional means, such as HPLCpurification.

The ordinarily skilled artisan will appreciate that factors such as pHand steric hindrance (i.e., the accessibility of the aldehyde tag toreaction with a reactive partner of interest) are of importance,Modifying reaction conditions to provide for optimal conjugationconditions is well within the skill of the ordinary artisan, and isroutine in the art. In general, it is normally desirable to conductionconjugation reactions at a pH below 7, with a pH of about 5.5, about 6,about 6.5, usually about 5.5 being optimal. Where conjugation isconducted with an aldehyde tagged polypeptide present in or on a livingcell, the conditions are selected so as to be physiologicallycompatible. For example, the pH can be dropped temporarily for a timesufficient to allow for the reaction to occur but within a periodtolerated by the cell having an aldehyde tag (e.g., from about 30 min to1 hour). Physiological conditions for conducting modification ofaldehyde tagged polypeptides on a cell surface can be similar to thoseused in a ketone-azide reaction in modification of cells bearingcell-surface azides (see, e.g., U.S. Pat. No. 6,570,040).

Small molecule compounds containing, or modified to contain, anα-nucleophilic group that serves as a reactive partner with an aldehydeof an FGly of an ald tag are also contemplated for use as drugs in thecarrier protein-drug conjugates of the present disclosure. Generalmethods are known in the art for chemical synthetic schemes andconditions useful for synthesizing a compound of interest (see, e.g.,Smith and March, March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; orVogel, A Textbook of Practical Organic Chemistry, Including QualitativeOrganic Analysis, Fourth Edition, New York: Longman, 1978).

Thus small molecules having an aminooxy or hydrazone group for reactionwith an aldehyde of an FGly of an ald-tagged carrier protein group areavailable or can be readily synthesized. An aminooxy or hydrazone groupcan be installed onto a small molecule using standard syntheticchemistry techniques. FIG. 12 provides a schematic of an exemplaryald-tagged carrier protein (represented by HSA) modified by conjugationto a small molecule drug (represented by doxorubicin).

Peptide Drug—Carrier Protein Conjugates

The conjugates of the present disclosure are site-specifically decoratedwith covalently bound drug. The site-specificity of reaction of areactive partner-containing drug with an aldehyde tag of the carrierprotein allows for production of carrier proteins having multiple sitesfor chemical conjugation, thus providing a scaffold for production ofcarrier protein-drug conjugates have a desired drug payload per proteinratio. Moreover, the relative position of the ald tags in the ald-taggedcarrier protein can be designed so as to provide for a desiredpresentation of covalently bound drug molecules on the surface of thefinal carrier protein-drug conjugate, thus allowing for control ofspatial orientation of the displayed drug payload.

Further, the site-specific nature of chemical modification of ald tagsto attach drug to the carrier protein can be exploited to provide for acomposition composed of a substantially homogenous population carrierprotein-drug conjugates. Such carrier protein-drug conjugates canprovide for control of the stoichiometry of drug delivery.

Carrier protein-drug conjugates of the present disclosure are composedof a carrier protein and one or more covalently bound drug molecules,where the carrier protein comprises a modified sulfatase motif of theformula:

X₁(FGly′)X₂Z₂X₃Z₃

where FGly′ is of the formula:

wherein J¹ is the covalently bound drug;

each L¹ is a divalent moiety independently selected from alkylene,substituted alkylene, alkenylene, substituted alkenylene, alkynylene,alkynylene, arylene, substituted arylene, cycloalkylene, substitutedcycloalkylene, heteroarylene, substituted heteroarylene, heterocyclene,substituted heterocyclene, acyl, amido, acyloxy, urethanylene,thioester, sulfonyl, sulfonamide, sulfonyl ester, —O—, —S—, —NH—, andsubstituted amine;

n is a number selected from zero to 40;

Z₂ is a proline or alanine residue;

X₁ is present or absent and, when present, is any amino acid, with theproviso that when the sulfatase motif is at an N-terminus of thepolypeptide, X₁ is present;

X₂ and X₃ are each independently any amino acid; and

Z₃ is a basic amino acid, and

wherein the carrier protein presents the covalently bound drug on asolvent-accessible surface when in a folded state. The X₁, X₂, Z₂, X₃,and Z₃ can be further defined as discussed above.

As noted above, the ald-tagged carrier protein can be designed so as toprovide for multiple sites for chemical conjugation, thus providing ascaffold for production of carrier protein-drug conjugates have adesired drug payload per protein ratio. The carrier protein-drugconjugates contemplated by the present disclosure generally include atleast 2 modified sulfatase motifs having covalently bound drugmolecules, and usually include 3 or more modified sulfatase motifshaving covalently bound drug molecules. The carrier protein-drugconjugates of the present disclosure can provide for a 4 or more, 5 ormore, or 6 or more covalently bound drug molecules in the carrierprotein-drug conjugate. Carrier protein-drug conjugates of the presentdisclosure thus include those having a drug payload to protein carrierratio of at least 2:1, at least 3:1, at least 4:1, at least 5:1 and,depending upon, for example, the size of the drug molecule relative tothe size of the carrier protein and/or the available sites for insertionof an ald tag on the solvent-accessible surface area of the foldedcarrier protein.

As noted above, the relative position of the ald tags in the ald-taggedcarrier protein can be designed so as to provide for a desiredpresentation of covalently bound drug molecules on the surface of thefinal carrier protein-drug conjugate. This feature allows for control ofspatial orientation of the displayed drug payload on the surface of thefinal carrier protein-drug conjugate. Carrier protein-drug conjugatescontaining multiple ald tags, which may include concatameric ald tagsseparated by flexible linkers as described herein, can provide forgreater drug payload:carrier protein ratios and enhanced presentation ofdrug to a physiological environment in which the carrier protein-drugconjugate is present. As such, the carrier protein-drug conjugates canbe described as a modified carrier protein “decorated” with drugcovalently bound to the carrier protein through an oxime or hydrazonelinkage to the peptide backbone of the carrier protein.

For example, the ald tags of the carrier protein-drug conjugate can bepositioned in the carrier protein-drug conjugate at at least one of anN-terminus of the carrier protein, a C-terminus of the carrier protein,and a solvent-accessible loop of the carrier protein. The ald tags canoptionally be provided in connection with a linker, e.g., a flexiblelinker, as described above. The multiple ald tags can be localized to aparticular region(s) of the carrier protein (e.g., provided in one ormore of a solvent-accessible loop, N-terminal region (includingN-terminus), C-terminal region (including C-terminus)), or can bedistributed over the solvent-accessible surface area of the foldedmodified carrier protein.

In general, it may be desirable to space the ald tags of the ald-taggedcarrier protein so that the final carrier protein-drug conjugate hascovalently bound drug spaced apart at a distance sufficient to avoidinteraction between the covalently bound drug molecules, e.g., so thatdrug molecules do not contact one another or otherwise interfere withtheir respective biological activities. The spatial orientation andpositioning within the carrier protein will vary according to a varietyof factors including the relative sizes of the drug to be conjugated andthe carrier protein. FIG. 10, bottom panel, provides a schematic of thethree-dimensional structure exemplifying an ald-tagged recombination HSAhaving a covalently bound GLP-1 peptide at its N-terminus.

As noted above, the site-specific nature of chemical modification of aldtags to attach drug to the carrier protein can be exploited to providefor a composition composed of a substantially homogenous populationcarrier protein-drug conjugates. Such carrier protein-drug conjugatescan provide for control of the stoichiometry of drug delivery. Suchhomogenous populations of carrier protein-drug conjugates include thosein which at least 60%, at least 70%, at least 80% at least 90% or moreof the carrier protein-drug conjugates of the population have the samedrug payload to carrier protein ratio.

Methods of Making Carrier Protein-Drug Conjugates

Methods of conjugation of an FGly-containing ald-tagged carrier proteinwith a reactive-partner containing-drug to provide a carrierprotein-drug conjugate having a desired drug payload:carrier proteinratio are contemplated by the present disclosure. In general, suchmethods involve combining an FGly-containing, ald-tagged carrier proteinwith a reactive partner-containing drug (e.g., an aminooxy- orhydrazide-containing drug) in a reaction mixture under conditionssuitable to promote reaction between the aldehyde(s) for the FGly(s) ofthe ald-tagged carrier protein with the reactive partner of the drugmolecule(s), thereby producing a reaction product of a carrierprotein-drug conjugate having drug covalently bound to the peptidebackbone of the carrier protein through an oxime bond, hydrazide bond,or other aldehyde specific chemistries such as reductive aminations, orWittig reactions.

After production of the ald-tagged carrier protein, it is isolated usingany of a variety of techniques available in the art (e.g.,chromatography, e.g., HPLC, FPLC, immunoaffinity purification, and thelike). In some embodiments, the carrier protein of the carrierprotein-drug conjugate contains an immunotag (e.g., His tag, FLAG tag),usually positioned at an N- or C-terminus to facilitate isolation andpurification prior to conjugation with drug. The FGly-containingald-tagged carrier protein for use in a conjugation reaction with drugcan be provided in denatured form or can be folder prior to combining inthe reaction mixture. Usually, the FGly-containing ald-tagged carrierprotein is provided in folded form in the conjugation reaction mixture.Where obtained from cells expressing the ald-tagged carrier protein anda compatible FGE, the FGly-containing ald-tagged carrier protein can beisolated in folded form from cells or, where secreted, from culturesupernatant. Where needed, methods for folding of proteins are availablein the art, and can be readily applied to the methods here.

In general, the ald-tagged carrier protein having FGly residues isisolated, and usually is purified. The carrier protein-drug conjugate iscombined in a reaction mixture in buffered solution with a reactivepartner-containing drug. The buffered solution can be at a physiologicalor near physiological pH, e.g., a pH of about 5 to 7, usually a pH ofabout 6.5. The reactive partner-containing drug is provided in thereaction mixture in excess to the aldehyde moieties of theFGly-containing ald-tagged carrier protein, usually at least 2 fold, 3fold, 4 fold, 5 fold or more excess, in order drive the reaction tocompletion. After addition of reactive partner-containing drug to thereaction mixture, the mixture is stirred under suitable conditions oftime and temperature (e.g., at room temperature for about 2 h). Theresulting carrier protein-drug conjugate is isolated from the reactionmixture and can be further purified using standard techniques (e.g.,chromatography, e.g., HPLC, FPLC).

Assessment of Carrier Protein-Drug Conjugate Activity

Following isolation of a carrier protein-drug conjugate from a reactionmixture, the carrier protein-drug conjugate can be screened for activityin one or more assays. Such assays can be for one or more biologicalactivities of the drug conjugated to the carrier protein-drug conjugateand/or for one or more characteristics of the carrier protein-drugconjugate (e.g., immunogenicity).

Methods for assessing immunogenicity are available in the art and can beadapted for use in assessing carrier protein-drug conjugates of thepresent disclosure. For example, the carrier protein-drug conjugate canbe administered to a non-human animal (e.g., an animal that can serve asa model for a human immune response), and the immune response to thecarrier protein-drug conjugate assessed. Carrier protein-drug conjugatescan be assessed for their activity in eliciting a humoral and/orcellular immune response in a non-human animal. Of particular interestis the production of anti-carrier protein-drug conjugate antibodies bythe immunized host. Methods for assessing antibody production in a hostare well known in the art.

Methods for assessing activity of the drug conjugated to the carrierprotein-drug conjugate are selected according to the drug bound to thecarrier protein-drug conjugate and are available in the art. Such assayscan be in vitro cell-free assays, in vitro cell-based assays, or in vivoassays (e.g., in an animal model). Usually the assay is a cell-based invitro functional assay or an in vivo assay using a non-human animalmodel (e.g, an animal model of human disease).

For example, activity of a carrier protein-GLP-1 conjugate of thepresent disclosure can be assayed in a cellular receptor activity assay,as exemplified in the Example below. Activity of a carrierprotein-calicitonin conjugate of the present disclosure can be assayedin a bone cell culture system to assess bone resorption of calcium.

Formulations

The carrier protein-drug conjugates of the present disclosure can beformulated in a variety of different ways. In general, the carrierprotein-drug conjugate is formulated in a manner compatible with thedrug conjugated to the carrier protein-drug conjugate, the condition tobe treated, and the route of administration to be used.

The carrier protein-drug conjugate can be provided in any suitable form,e.g., in the form of a pharmaceutically acceptable salt, and can beformulated for any suitable route of administration, e.g., oral, topicalor parenteral administration. Where the carrier protein-drug conjugateis provided as a liquid injectable (such as in those embodiments wherethey are administered intravenously or directly into a tissue), thecarrier protein-drug conjugate can be provided as a ready-to-use dosageform, or as a reconstitutable storage-stable powder or liquid composedof pharmaceutically acceptable carriers and excipients.

Methods for formulating carrier protein-drug conjugates can be adaptedfrom those available in the art. For example, carrier protein-drugconjugates can be provided in a pharmaceutical composition comprising aneffective amount of a carrier protein-drug conjugate and apharmaceutically acceptable carrier (e.g., saline). The pharmaceuticalcomposition may optionally include other additives (e.g., buffers,stabilizers, preservatives, and the like). Of particular interest areformulations that are suitable for administration to a mammal,particularly those that are suitable for administration to a human.

Methods of Treatment

The carrier protein-drug conjugates of the present disclosure find usein treatment of a condition or disease in a subject that is amenable totreatment by administration of the parent drug (i.e., the drug prior toconjugation to the carrier protein. By “treatment” is meant that atleast an amelioration of the symptoms associated with the conditionafflicting the host is achieved, where amelioration is used in a broadsense to refer to at least a reduction in the magnitude of a parameter,e.g. symptom, associated with the condition being treated. As such,treatment also includes situations where the pathological condition, orat least symptoms associated therewith, are completely inhibited, e.g.,prevented from happening, or stopped, e.g. terminated, such that thehost no longer suffers from the condition, or at least the symptoms thatcharacterize the condition. Thus treatment includes: (i) prevention,that is, reducing the risk of development of clinical symptoms,including causing the clinical symptoms not to develop, e.g., preventingdisease progression to a harmful state; (ii) inhibition, that is,arresting the development or further development of clinical symptoms,e.g., mitigating or completely inhibiting an active disease; and/or(iii) relief, that is, causing the regression of clinical symptoms.

The subject to be treated can be one that is in need of therapy, wherethe host to be treated is one amenable to treatment using the parentdrug. Accordingly, a variety of subjects may be amenable to treatmentusing the carrier protein-drug conjugates disclosed herein. Generallysuch subjects are “mammals”, with humans being of particular interest.Other subjects can include domestic pets (e.g., dogs and cats),livestock (e.g., cows, pigs, goats, horses, and the like), rodents(e.g., mice, guinea pigs, and rats, e.g., as in animal models ofdisease), as well as other primates (e.g., chimpanzees, and monkeys.

The amount of carrier protein-drug conjugate administered can beinitially determined based on guidance of a dose and/or dosage regimenof the parent drug. In general, the carrier protein-drug conjugates canprovide for enhanced blood serum half-life of the bound drug, thusproviding for at least one of reduced dose or reduced administrations ina dosage regimen. Thus the carrier protein-drug conjugates can providefor reduced dose and/or reduced administration in a dosage regimenrelative to the parent drug prior to conjugated in a carrierprotein-drug conjugate of the present disclosure.

Furthermore, as noted above, because the carrier protein-drug conjugatescan provide for controlled stoichiometry of drug delivery, dosages ofcarrier protein-drug conjugates can be calculated based on the number ofdrug molecules provided on a per carrier protein-drug conjugate basis.

Accordingly, the carrier protein-drug conjugates of the presentdisclosure where in the drug is GLP-1, or a biologically active variantthereof, can be used in treatment of conditions amenable to therapy byadministration of GLP-1. Such conditions include Type II diabetes andhyperglycemia. Such methods involve administration of an effectiveamount of a carrier protein-GLP-1 conjugate (or a carrier protein-drugconjugate having a covalently bound variant of GLP-1) to a subject inneed to treatment (e.g., a subject having or at risk of Type II diabetesand/or hyperglycemia, wherein administration of the carrier protein-drugconjugate is effect to treat the condition.

Where the drug of the carrier protein-drug conjugates of the presentdisclosure is Calcitonin, or a biologically active variant thereof, canbe used in treatment of conditions amenable to therapy by administrationof Calcitonin Such conditions include osteoporosis and hypercalcaemia.Such methods involve administration of an effective amount of a carrierprotein-Calcitonin conjugate (or a carrier protein-drug conjugate havinga covalently bound variant of Calcitonin) to a subject in need totreatment (e.g., a subject having or at risk of osteoporosis orhypercalcaemia, wherein administration of the carrier protein-drugconjugate is effect to treat the condition.

Kits and Systems

Kits and systems are provided to facilitate and, where desired,standardize the compositions of the invention and the uses thereof. Kitscontemplated herein can include one or more of a construct encoding analdehyde tagged carrier protein (and may encompass a library composed ofconstructs encoding a population of differently ald-tagged carrierproteins) for expression in a host cell; a host cell that produces anFGE compatible with an aldehyde tag of the kit, where the FGE may beendogenous, recombinant, or heterologous; a host cell geneticallymodified to express an aldehyde tagged carrier protein (and mayencompass a library composed of recombinant host cells containingconstructs encoding a population of differently ald-tagged carrierproteins), which host cell can further express an endogenous,recombinant, or heterologous FGE compatible for conversion of thealdehyde tag of the tagged polypeptide; reagents to provide forproduction of a reactive partner-containing drug; and reagents topromote a reaction between an ald-tagged carrier protein and a reactivepartner-containing drug.

In addition, the kit can contain instructions for using the componentsof the kit, particularly the compositions of the invention that arecontained in the kit.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Production of Panel of HSA Carrier Proteins

The ald-tag technology is used to provide for high-efficiencymodification of secreted carrier proteins in mammalian cell culturesystem. An FGE and sulfatase motifs are used to install aldehyde tags ina recombinant human serum albumin (rHSA) in a yeast expression system,e.g., Saccharomyces cerevisiae. The FGly-containing ald tag will beexploited to conjugate drugs (e.g., small molecule drugs) to the HSAcarrier protein. HSA is trafficked through the secretory pathway,similar to native sulfatases and the previously studied secreted Fcproteins, and are therefore will be readily be recognized as substratesby ER-resident FGE when expressed in mammalian cells. When expressed inyeast, the recombinant HSA containing the aldehyde tag motif is purifiedand reacted with purified recombinant FGE to convert the cysteine toformylglycine ex vivo, the addition of purified FGE to a purifiedrecombinant protein to give the enzymatic transformation. The convertedaldehyde tagged HSA were chemically modified with small molecules toafford a final conjugated protein construct.

C-Terminal Modified HSA as a Model Protein

The vector construction utilized the native (human) HSA leader sequence.Modifications to the sequence were made after initial cloning of HSAinto a plasmid. A strong promoter was used (for example, Galactoseinducible GAL or constitutively-active GPD promoter with -LEU2. Forexample, the vector p425-GAL1 or the vector p425GalL have agalactose-inducible strong promoter in place with a MCS that can receivethe HSA sequence.

Yeast cell lines containing the HSA-Ald₆ tag construct were generatedusing standard molecular biology techniques. After establishing andconfirming transformed colonies by PCR, the expression of the Ald₆tagged-HSA was induced and production assayed by immunoblotting. Inorder to probe directly the aldehyde-modified protein, rHSA was reactedwith aminooxy-FLAG peptide and analyzed by Western blot with ananti-FLAG antibody as well as an anti-HSA antibody. The percentconversion of Cys to FGly was quantified by isolation of the proteinfollowed by tryptic digestion and mass spectrometry analysis.

Example 2 Production of Carrier Proteins Having Multiple Ald Tags

Carrier proteins conjugated with multiple peptide drugs or smallmolecules can greatly enhance the efficacy of the biotherapeutic ofinterest. Thus multiple aldehyde tags will be installed into a singleHSA carrier protein. An HSA carrier protein having Ald₆ tags (LCTPSR,(SEQ ID NO:1)) placed in different locations along the peptide backboneof the carrier protein will be generated.

Three different Ald₆ tag sequences were appended to the recombinant HSAcarrier protein. These constructs, containing Ald₆ sequences, are shownin FIG. 6 (depicting three uniquely tagged proteins). The constructswere expressed in S. cerevisae, purified and reacted along with purifiedM. tuberculosis FGE. Reaction conditions were optimized to maximize theconversion of the cysteine to FGly. The converted protein was purifiedand analyzed for the presence of FGly by reaction with aminooxy-biotinor aminooxy FLAG peptide followed by Western blot. A CHO expressionsystem was developed for the production of HSA and E. coli expressionsystems can also be developed for production of ald-tagged rHSAs. Thepercent conversion of Cys to FGly for each individual tag in the seriesof multi-ald-tagged rHSAs is assessed by isolation of the proteinfollowed by tryptic digestion and mass spectrometry analysis.

A panel of ald-tagged rHSA carrier proteins was generated, withdifferently ald-tagged rHSA carrier proteins having aldehydes placedstrategically throughout the scaffold. The panel thus serves as alibrary of differently ald-tagged rHSAs, where the members of thelibrary differ in the number and/or position of ald tags in thescaffold. For example, as exemplified in FIG. 1B, one simple panel wasgenerated where the members include an ald-tagged rHSA having an ald-tagposition at the N-terminus, an ald-tagged rHSA having an ald-tagposition at the C-terminus, an ald-tagged rHSA having an ald-tagpositioned within a loop of the protein. Another library includes thesemembers, as well as ald-tagged rHSAs having ald tags at both the N- andC-termini, at the N-termini and a solvent-accessible loop, at theC-termini and a solvent-accessible loop, and at each of the N-termini,the C-termini and a solvent-accessible loop. FIG. 10 provides thethree-dimensional structure of HSA, which can be used for guidance inselecting sites for ald-tag insertion. Exemplary carrier protein-drugconjugates of HSA having a peptide drug positioned at exemplarysolvent-accessible sites on HSA are provided in the bottom portion ofFIG. 10.

Example 3 Conjugation of Peptides to the Protein Scaffold

Short serum half-life has been a challenge in the development peptidetherapeutics. Peptides are typically cleared from the bloodstream withinminutes to hours after administration, and thus may not be sufficientlyexposed in the target tissue for a desired clinical effect. Aldehydetagged carrier proteins, such as ald tagged HSA can be used as a carrierprotein to increase the serum half-life of the peptides.

Two carrier protein-drug conjugates are generated—one a conjugate withCalcitonin and one a conjugate with GLP-1. As mentioned previously, bycoupling the peptides to the HSA carrier protein the absorption andelimination half-lives will be increased.

The peptides were synthesized via standard Fmoc-based solid phasepeptide synthesis protocols. The final residue added at the N terminuswas (t-Boc-aminooxy)acetic acid followed by cleavage under standardconditions. Deprotection to expose the amino-oxy functionality isfollowed by HPLC purification. Purified ald-tagged HSA is added to abuffered solution of peptide that has been functionalized with anN-terminus amino-oxy functionality. Upon coupling to the ald-tagged HSA,the final protein-peptide complex is purified using FPLC.

Example 4 Assessment of HSA-GLP-1 Conjugates

The HSA-GLP-1 conjugate is assayed for activity as compared to nativeGLP-1. GLP-1, released from intestinal L-cells, is known for its potentstimulation of insulin biosynthesis and release from pancreatic(3-cells. For the identification of GLP-1 receptor agonist, a cellularreceptor activation assay based on the formation of cAMP occurring dueto receptor activation is used. Receptor activation studies areperformed by incubating RINm5F cells, a rat insulinoma cell line, withor without the test peptides or the HSA-peptide conjugates at increasingconcentrations. Activation of the GLP-1 receptor is measured byquantification of the intracellular cAMP after cell lysis. EC₅₀ values(concentration of test compound leading to a half maximal stimulation ofcamp production) are calculated from the resulting dose response curves.

Example 5 Assessment of HSA-Calcitonin Conjugates

The HSA-Calcitonin conjugate is assayed for osteoclast activity ascompared to native Calcitonin. The BD BioCoat Osteologic Bone CellCulture System is used to assess the effect of treatment with theHSA-peptide conjugate and the native Calcitonin peptide on boneresorption of calcium. The BD BioCoat Osteologic Bone Cell CultureSystem involves sub-micron synthetic calcium phosphate thin films coatedonto various culture vessels. This system has been used as analternative method for compound screening for direct assessment ofosteoclast and osteoblast activity in vitro. The thin film designpermits easy and reliable quantification of results.

Example 6 Construction of Aldehyde Tagged Human Serum Albumin CarrierProteins

The following describes production of an exemplary ald-tagged HSA.

A. Primer Design:

A nucleic acid encoding the wildtype HSA was inserted into a vectorwhich can be exploited for as a template for subcloning. Using a vectorwith the appropriate internal restriction sites the first PCR productwas:

-xmaI-----HSA-stop_codon-----xhoI-

After insertion of this sequence in a vector, variants were made usinglonger primers such as:

-xmaI-----HSA-6xhis-stop_codon-----xhoI-

-xmaI-----HSA-LCTPSR-stop_codon-----xhoI

The following primers were used for PCR cDNA amplification.

Forward: (SEQ ID NO: 98) 5′-AATCCCGGG ATGAAGTGGGTAACCTTTATTTCCC-3′Reverse: (SEQ ID NO: 99) 5′-TGACTCGAG TTATAAGCCTAAGGCAGCTTGACTTG-3′

The double underline represents the native sequence, with the singleunderline the newly introduced restriction sites for further cloning.

A 1830 bp fragment was isolated after gel purification. This wasfollowed by digestion with XmaI and XhoI, and the DNA fragment insertedin the expression vector. The open reading frame for HSA in theexpression vector was as follows.

HSA-Encoding Nucleic Acid Sequence:

(SEQ ID NO: 100) aatcccgggatgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgatcgtcgagatgcacacaagagtgaggttgctcatcggataaagatttgggagaagaaaatttcaaagccaggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgatttcatgacaatgaagagacatattgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgagccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccagccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgacgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctattgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcagtagaggtctcaagaaacctaggaaaagtgggcagcaaatgagtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgagcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccaccatgcagatatatgcacactactgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttaactcgag

Amino Acid Sequence of Encoded HSA

The HSA native leader sequence (single and double underlined residues)is removed in 2-step process (in humans) before secretion of matureprotein:

(SEQ ID NO: 101) NPGMKWVTFI SLLFLFSSAY S RGVFRRDAH KSEVAHRFKDLGEENFKALV LIAFAQYLQQ CPFEDHVKLV NEVTEFAKTCVADESAENCD KSLHTLFGDK LCTVATLRET YGEMADCCAKQEPERNECFL QHKDDNPNLP RLVRPEVDVM CTAFHDNEETFLKKYLYEIA RRHPYFYAPE LLFFAKRYKA AFTECCQAADKAACLLPKLD ELRDEGKASS AKQRLKCASL QKFGERAFKAWAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLECADDRADLAKY ICENQDSISS KLKECCEKPL LEKSHCIAEVENDEMPADLP SLAADFVESK DVCKNYAEAK DVFLGMFLYEYARRHPDYSV VLLLRLAKTY ETTLEKCCAA ADPHECYAKVFDEFKPLVEE PQNLIKQNCE LFEQLGEYKF QNALLVRYTKKVPQVSTPTL VEVSRNLGKV GSKCCKHPEA KRMPCAEDYLSVVLNQLCVL HEKTPVSDRV TKCCTESLVN RRPCFSALEVDETYVPKEFN AETFTFHADI CTLSEKERQI KKQTALVELVKHKPKATKEQ LKAVMDDFAA FVEKCCKADD KETCFAEEGK KLVAASQAAL GLTR

B. Construction of C-Terminal Modified HSA

Using the plasmid with native HSA as a PCR template a new 3′ HSA PCRprimer with additional restriction sites for appending C-terminal tagsonto recombinant HSA was designed as follows:

(SEQ ID NO: 102) 5′-ATACTCGAG TTA GTCGACTTCAAGCTT TAAGCCTAAGGCAGCTTGACTTG-3′

Double underline: native C-terminus of HSA sequence.

Single underline adjacent double underline: HinDIII site

Bold residues=Stop codon

Single underline 3′ of stop codon: SalI site

Single underline 5′ of stop codon: XhoI site

The SalI and HindIII were provided in the primer as these are not in theplasmid constructs. Used in conjunction with the same Forward primerused for original HSA amplification from cDNA, an 1863 residue PCRproduct was obtained as follows (with the predicted amino sequencefollowing):

(SEQ ID NO: 103)aatcccgggatgaagtgggtaaccatatacccttctattctctttagctcggcttattccaggggtgtgatcgtcgagatgcacacaagagtgaggagctcatcggtttaaagatagggagaagaaaatttcaaagccaggtgagattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctattggagacaaattatgcacagagcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcacttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcattcatgacaatgaagagacatttagaaaaaatacttatatgaaattgccagaagacatccttactatatgccccggaactccattattgctaaaaggtataaagctgctatacagaatgagccaagctgctgataaagctgcctgcctgagccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtaccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccagccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattagttgaaagtaaggatgatgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgatagtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgacgatgaatttaaacctcagtggaagagcctcagaatttaatcaaacaaaattgtgagctattgagcagcaggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcagtagaggtctcaagaaacctaggaaaagtgggcagcaaatgagtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgagcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccaggtgaacaggcgaccatgctatcagctctggaagtcgatgaaacatacgacccaaagagataatgctgaaacattcaccaccatgcagatatatgcacactactgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctagccgaggagggtaaaaaacttgagctgcaagtcaagctgccttaggcttaaagcttgaagtcgactaactcgag ata(SEQ ID NO: 104) NPG MKWVTFI SLLFLFSSAY S RGVFRRDAH KSEVAHRFKD LGEENFKALV LIAFAQYLQQCPFEDHVKLV NEVTEFAKTC VADESAENCD KSLHTLFGDK LCTVATLRET YGEMADCCAKQEPERNECFL QHKDDNPNLP RLVRPEVDVM CTAFHDNEET FLKKYLYEIA RRHPYFYAPELLFFAKRYKA AFTECCQAAD KAACLLPKLD ELRDEGKASS AKQRLKCASL QKFGERAFKAWAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLEC ADDRADLAKY ICENQDSISSKLKECCEKPL LEKSHCIAEV ENDEMPADLP SLAADFVESK DVCKNYAEAK DVFLGMFLYEYARRHPDYSV VLLLRLAKTY ETTLEKCCAA ADPHECYAKV FDEFKPLVEE PQNLIKQNCELFEQLGEYKF QNALLVRYTK KVPQVSTPTL VEVSRNLGKV GSKCCKHPEA KRMPCAEDYLSVVLNQLCVL HEKTPVSDRV TKCCTESLVN RRPCFSALEV DETYVPKEFN AETFTFHADICTLSEKERQI KKQTALVELV KHKPKATKEQ LKAVMDDFAA FVEKCCKADD KETCFAEEGKKLVAASQAAL GLKLEVDLEI

The product was digested with XmaI and XhoI and inserted into theexpression vector, then digested with HinDIII and SalI (sequentially)for insertion of a synthetic piece of double-stranded DNA withcomplementary sticky ends. The sequence of the synthetic DNA encodingthe HSA having an ald tag LCTPSR (SEQ ID NO:1) at the C terminus isprovided below (with the predicted amino sequence following):

(SEQ ID NO: 105)aacccgggcatgaaatgggtgacctttattagcctgctgtttctgtttagcagcgcgtatagccgcggcgtgtttcgccgcgatgcgcataaaagcgaagtggcgcatcgctttaaagatctgggcgaagaaaactttaaagcgctggtgctgattgcgtttgcgcagtatctgcagcagtgcccgatgaagatcatgtgaaactggtgaacgaagtgaccgaatttgcgaaaacctgcgtggcggatgaaagcgcggaaaactgcgataaaagcctgcataccctgtaggcgataaactgtgcaccgtggcgaccctgcgcgaaacctatggcgaaatggcggattgctgcgcgaaacaggaaccggaacgcaacgaatgctactgcagcataaagatgataacccgaacctgccgcgcctggtgcgcccggaagtggatgtgatgtgcaccgcgatcatgataacgaagaaacctactgaaaaaatatctgtatgaaattgcgcgccgccatccgtattatatgcgccggaactgctgttttttgcgaaacgctataaagcggcgtttaccgaatgctgccaggcggcggataaagcggcgtgcctgctgccgaaactggatgaactgcgcgatgaaggcaaagcgagcagcgcgaaacagcgcctgaaatgcgcgagcctgcagaaatttggcgaacgcgcgtttaaagcgtgggcggtggcgcgcctgagccagcgctttccgaaagcggaatttgcggaagtgagcaaactggtgaccgatctgaccaaagtgcataccgaatgctgccatggcgatctgctggaatgcgcggatgatcgcgcggatctggcgaaatatatttgcgaaaaccaggatagcattagcagcaaactgaaagaatgctgcgaaaaaccgctgctggaaaaaagccattgcattgcggaagtggaaaacgatgaaatgccggcggatctgccgagcctggcggcggattttgtggaaagcaaagatgtgtgcaaaaactatgcggaagcgaaagatgtgtttctgggcatgtttctgtatgaatatgcgcgccgccatccggattatagcgtggtgctgctgctgcgcctggcgaaaacctatgaaaccaccctggaaaaatgctgcgcggcggcggatccgcatgaatgctatgcgaaagtgatgatgaatttaaaccgctggtggaagaaccgcagaacctgattaaacagaactgcgaactgatgaacagctgggcgaatataaatttcagaacgcgctgctggtgcgctataccaaaaaagtgccgcaggtgagcaccccgaccctggtggaagtgagccgcaacctgggcaaagtgggcagcaaatgctgcaaacatccggaagcgaaacgcatgccgtgcgcggaagattatctgagcgtggtgctgaaccagctgtgcgtgctgcatgaaaaaaccccggtgagcgatcgcgtgaccaaatgctgcaccgaaagcctggtgaaccgccgcccgtgctttagcgcgctggaagtggatgaaacctatgtgccgaaagaatttaacgcggaaacctttacctttcatgcggatatttgcaccctgagcgaaaaagaacgccagattaaaaaacagaccgcgctggtggaactggtgaaacataaaccgaaagcgaccaaagaacagctgaaagcggtgatggatgattagcggcgtagtggaaaaatgctgcaaagcggatgataaagaaacctgctttgcggaagaaggcaaaaaactgctgtgcaccccgagccgcgtggatctggaaatt (SEQ ID NO: 106) NPG MKWVTFI SLLFLFSSAY S RGVFRRDAH KSEVAHRFKD LGEENFKALV LIAFAQYLQQCPFEDHVKLV NEVTEFAKTC VADESAENCD KSLHTLFGDK LCTVATLRET YGEMADCCAKQEPERNECFL QHKDDNPNLP RLVRPEVDVM CTAFHDNEET FLKKYLYEIA RRHPYFYAPELLFFAKRYKA AFTECCQAAD KAACLLPKLD ELRDEGKASS AKQRLKCASL QKFGERAFKAWAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLEC ADDRADLAKY ICENQDSISSKLKECCEKPL LEKSHCIAEV ENDEMPADLP SLAADFVESK DVCKNYAEAK DVFLGMFLYEYARRHPDYSV VLLLRLAKTY ETTLEKCCAA ADPHECYAKV FDEFKPLVEE PQNLIKQNCELFEQLGEYKF QNALLVRYTK KVPQVSTPTL VEVSRNLGKV GSKCCKHPEA KRMPCAEDYLSVVLNQLCVL HEKTPVSDRV TKCCTESLVN RRPCFSALEV DETYVPKEFN AETFTFHADICTLSEKERQI KKQTALVELV KHKPKATKEQ LKAVMDDFAA FVEKCCKADD KETCFAEEGK KLLCTPSR VD LEI

The plasmid encoding recombinant HSA was further modified to include theFGE motif at the C-terminus. Primers, designed for the insertion of FGEmotif and thrombin-cleavable affinity tag at C-terminus, were ligatedinto the vector using standard molecular biology techniques. The insertdesign was as follows:

(SEQ ID NO:: 107) HinDIII-L C TPSR-LVPRGS-PstI-HHHHHH-SalI(SEQ ID NO: 8) 5′ AGCTTCTT TGT ACCCCTAGCAGGCTGGTGCCGCGCGGCAGCCTGCAGCATCATCACCACCATCACG (SEQ ID NO: 109) 5′ AGAA ACATGGGGATCGTCCGACCACGGCGCGCCGTCGGACGTCGTAG TAGTGGTGGTAGTGCAGCT

PstI site allows for detection of insert via diagnostic digestioninstead of sequencing each miniprep. LVPRGS is a thrombin cleavage site.The ORF translates to:

(SEQ ID NO: 110) MKWTFISLLF LFSSAYSRGV FRRDAHKSEV AHRFKDLGEENFKALVLIAF AQYLQQCPFE DHVKLVNEVT EFAKTCVADESAENCDKSLH TLFGDKLCTVA TLRETYGEMA DCCAKQEPERNECFLQHKDD NPNLPRLVRP EVDVMCTAFH DNEETFLKKYLYEIARRHPY FYAPELLFFAK RYKAAFTECC QAADKAACLLPKLDELRDEGK ASSAKQRLKCA SLQKFGERA FKAWAVARLSQRFPKAEFAEV SKLVTDLTKV HTECCHGDLL ECADDRADLAKYICENQDSI SSKLKECCEK PLLEKSHCIA EVENDEMPADLPSLAADFVE SKDVCKNYAE AKDVFLGMFL YEYARRHPDYSVVLLLRLAK TYETTLEKCC AAADPHECYAK VFDEFKPLVEEPQNLIKQNCE LFEQLGEYKFQ NALLVRYTKK VPQVSTPTLVEVSRNLGKVG SKCCKHPEAK RMPCAEDYLS VVLNQLCVLHEKTPVSDRVT KCCTESLVNR RPCFSALEVD ETYVPKEFNAETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKEQLKAVMDDFAAF VEKCCKADDK ETCFAEEGKK LVAASQAALG LKL

LV PRGSLQHHHH HHVD

The construct was expressed in S. cerevisae and purified using affinitycolumn purification (FIG. 13). The modified protein was reacted withpurified FGE to convert the cysteine to formyl glycine. The recombinantHSA was reacted with a fluorophore containing a hydrazide and theconversion and conjugation was quantified by measuring the resultingfluorescence of the modified protein (FIG. 13).

C. Construction of N-Terminal Modified HSA

Generating the N-terminal aldehyde tagged HSA was accomplished byinserting an in-frame synthetic gene where the N-terminus of mature HSAwas modified with the aldehyde tag. The synthetic gene was cloned into ayeast expression vector using standard molecular biology techniques. Thedesigned sequence is as follows. Bold/underline=restriction site arrays

aaacgatg = kozak (shine dalgarno) sequenceaagtgggtaacctttatttcccttctattctctttagctcggcttattccaggggtgtgtttcgtcga(SEQ ID NO: 11) = prepro region (removed from mature protein)caccatcatcaccaccatcac (SEQ ID NO: 112) = 7xHIS tagctggtgccgcgcggcagc (SEQ ID NO: 113) = thrombin recognition sitectttgtacccctagcagg (SEQ ID NO: 114) = LCTPSR motif (SEQ ID NO: 1)ggaggc = diglycine linker taa = stop codon (SEQ ID NO: 115) aaataaagcttcccgggggatcc aaacgatgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcacacaagcaccatcatcaccaccatcacctggtgccgcgcggcagcctttgtacccctagcaggggaggcagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgagccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagaccttaccaaagtccacacggaatgctgccatggagacctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttataat gaattcgtcgacctcgaggatatc acaag

The expected ORF product was

(SEQ ID NO: 116) MKWVTFISLL FLFSSAYSRG VFRRDAHKHH HHHHHLVPRG S LCTPSRGGS EVAHRFKDLG EENFKALVLI AFAQYLQQCPFEDHVKLVNE VTEFAKTCVA DESAENCDKS LHTLFGDKLCTVATLRETYG EMADCCAKQE PERNECFLQH KDDNPNLPRLVRPEVDVMCT AFHDNEETFL KKYLYEIARR HPYFYAPELLFFAKRYKAAF TECCQAADKA ACLLPKLDEL RDEGKASSAKQRLKCASLQK FGERAFKAWA VARLSQRFPKA EFAEVSKLVTDLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLKECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVCKNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETTLEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFEQLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSKCCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKCCTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTLSEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVEKCCKADDKET CFAEEGKKLV AASQAALGL

The construct was expressed in S. cerevisae and purified using affinitycolumn purification. The modified protein was reacted with purified FGEto convert the cysteine to formylglycine. The recombinant HSA wasreacted with a fluorophore containing a hydrazide and the conversion andconjugation was quantified by measuring the resulting fluorescence ofthe modified protein.

D. Construction of Internal Modified HSA

Generating the internal aldehyde tagged HSA was accomplished byinserting an in-frame synthetic gene where key restriction sites areplaced where mature HSA is to be modified with the aldehyde tag. Thesynthetic gene was cloned into a yeast expression vector using standardmolecular biology techniques. The designed sequence was as follows:

(SEQ ID NO: 117) CGAAGGATCCAAACGATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAGGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACTGACCTTACCAAAGTCCACACGGAATGCTGTCACGGAGACCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTCTCGAGCCTTCTACTAGTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACACTTGAGAAGTGCTGTGCCGCCGCTGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTACCCGGGTCTACTCCGCGGCTGGTGCCGCGCGGCAGCCTTCAACATCATCACCACCATCACGTCGACTAA TGGAATTCCCTA

The expected ORF was:

(SEQ ID NO: 118) MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGEENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLKKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAVARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPALEPSTS ADFVESKDVC KNYAEAKDVF LGMFLYEYARRHPDYSVVLL LRLAKTYETTL EKCCAAADPH ECYAKVFDEFKPLVEEPQNL IKQNCELFEQ LGEYKFQNAL LVRYTKKVPQVSTPTLVEVS RNLGKVGSKC CKHPEAKRMP CAEDYLSVVLNQLCVLHEKT PVSDRVTKCC TESLVNRRPC FSALEVDETYVPKEFNAETF TFHADICTLS EKERQIKKQT ALVELVKHKPKATKEQLKAV MDDFAAFVEK CCKADDKETC FAEEGKKLVAASQAALGLPG STPRLVPRGS LQHHHHHHVD

6xHis-HSA synthetic gene was ligated into pCR blunt II-TOPO vector,followed by digestion of pRW33 with EcoRI and BamHI to cut out6xHis-HSA, which was purified and then ligated into pcDNA3.1 using theEcoRI and BamHI sites. The resulting vector was Digested with XhoI/SpeIand the annealed primers:

(SEQ ID NO: 119) 5′-CTAGCCTTTGTACCCCTAGCAGGG-3′ and (SEQ ID NO: 120)5-CTAGCCCTGCTAGGGGTACAAAGA-3′

were ligated in generating the aldehyde tag. The designed sequence wasas follows:

(SEQ ID NO: 121) CCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAGGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACTGACCTTACCAAAGTCCACACGGAATGCTGTCACGGAGACCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTCTCGATCTTTGTACCCCTAGCAGGGCTACTAGTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACACTTGAGAAGTGCTGTGCCGCCGCTGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTACCCGGGTCTACTCCGCGGCTGGTGCCGCGCGGCAGCCTTCAACATCATCACCACCATCACGTCGACTAATGGAATTCCCTA

The expected ORF product was:

(SEQ ID NO: 122) MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGEENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLKKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAVARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND EMPALD LCTP SRATSADFVE SKDVCKNYAE AKDVFLGMFLYEYARRHPDY SVVLLLRLAK TYETTLEKCC AAADPHECYAKVFDEFKPLV EEPQNLIKQN CELFEQLGEY KFQNALLVRYTKKVPQVSTP TLVEVSRNLG KVGSKCCKHP EAKRMPCAEDYLSVVLNQLC VLHEKTPVSD RVTKCCTESL VNRRPCFSALEVDETYVPKE FNAETFTFHA DICTLSEKER QIKKQTALVELVKHKPKATK EQLKAVMDDF AAFVEKCCKA DDKETCFAEEGKKLVAASQA ALGLPGSTPR LVPRGSLQHH HHHHVD

6xHis-LCTPSR-HSA was expressed and purified from CHO cells.6xHis-LCTPSR-HSA was transfected in pcDNA3.1 into CHO cells in Opti-MEMserum-free medium using Lipofectin transfection reagent in a 10 cm dish.After 3 h at 37° C., the Opti-MEM medium was removed and added 10 mL ofHAM'S F12 serum-free medium. After 3 days at 37°, the media wascollected and added 10 mL Binding Buffer (20 mM Na₂PO₄, 500 mM NaCl, 20mM Imidazole, pH 7.5) and 200 μl of Ni-NTA resin. After incubating withrotation for 1 h at 4° C., the mixture was applied to a column. Theresin was washed with 5 mL Binding Buffer and then eluted with 5×500 μlElution Buffer (20 mM Na₂PO₄, 500 mM NaCl, 500 mM Imidazole, pH 7.5).The samples were run on 10% Tric-HCl gels and either stained withCoomassie or transferred to nitrocellulose for immunoblotting with ananti-His antibody to verify the presence of protein.

E. Construction HSA Modified with Two Aldehyde Tags, Internally Modifiedand C-Terminally Modified HSA

The vectors containing recombinant HSA was digested with XmaI/SacII andthe annealed primers 5′-CCGGACTTTGTACCCCTAGCAGGGGGC-3′ (SEQ ID NO:123)and 5′-CCCCTGCTAGGGGTACAAAGT-3′ (SEQ ID NO:124) were ligated inresulting in the insertion of the aldehyde tag. The designed sequencewas as follows:

(SEQ ID NO: 125) GAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAGGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACTGACCTTACCAAAGTCCACACGGAATGCTGTCACGGAGACCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTCTCGATCTTTGTACCCCTAGCAGGGCTACTAGTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACACTTGAGAAGTGCTGTGCCGCCGCTGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTACCCGGACTTTGTACCCCTAGCAGGGGGCGGCTGGTGCCGCGCGGCAGCCTTCAACATCATCACCACCATCACGTCGACTAATGGAATTCCCTA

The expected ORF product was:

(SEQ ID NO: 126) MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGEENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLKKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAVARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPALDLCTP SRATSADFVE SKDVCKNYAE AKDVFLGMFLYEYARRHPDY SVVLLLRLAK TYETTLEKCC AAADPHECYAKVFDEFKPLV EEPQNLIKQN CELFEQLGEY KFQNALLVRYTKKVPQVSTP TLVEVSRNLG KVGSKCCKHP EAKRMPCAEDYLSVVLNQLC VLHEKTPVSD RVTKCCTESL VNRRPCFSALEVDETYVPKE FNAETFTFHA DICTLSEKER QIKKQTALVELVKHKPKATK EQLKAVMDDF AAFVEKCCKA DDKETCFAEEGKKLVAASQA ALGLPGLCTP SRGRLVPRGS LQHHHHHHV D

Example 7 Production of Drug-HSA Conjugate

Purified HSA modified with aldehyde tags was added to a bufferedsolution of peptide that has been functionalized with an N-terminusamino-oxy functionality. The conjugation reaction is carried out in abuffered solution at a pH of 6.0 with 100 mM aniline added. A three-foldexcess of aminooxy peptide to aldehyde was added to the reaction mixtureto drive the reaction to completion. After addition of peptide to asolution of the ald-tagged HSA, the mixture was stirred at room temp for2 h, dialyzed and the protein-peptide conjugate purified using FPLC.

Example 8 Expression of Aldehyde-Tagged HSA with Pichia Pastoris

6xHis-LCTPSR-HSA was transformed into the Pichia strain GS 115 usingpRW39 (6xHis-LCTPSR-HSA in pPIC3.5K). pRW39 was linearized with BglII.20 μg of DNA in 10 μL water was added to 80 μL of freshly competentGS115 cells and electroporated in a 2 mm cuvette (2000V). 1 mL of icecold 1M sorbitol was added immediately after electroporation. The cellswere plated on regeneration dextrose Bacto agar plates (lackinghistidine to select for HIS+ transformants) and incubated 30° C. for 3days. Colonies were isolated and tested for resistance to G418 to selectfor those colonies containing multiple copies of aldehyde-tagged-HSAintegrated into the Pichia genome.

Colonies were grown 10 mL of buffered buffered glycerol-complex mediumovernight at 30° C. Cultures were centrifuged the cells were resuspendedin buffered methanol-complex medium to induce expression of6xHis-LCTPSR-HSA, integrated into the Pichia genome under the control ofa methanol-inducible promoter. The cells were grown for 6 days at 30° C.Methanol was added to each culture every 24 h to 0.5%. After 6 days,cells were cleared from the media by centrifugation and 10 μL of themedia was run on an SDS-PAGE gel, and the gel was stained with CoomassieBlue. As a negative control, the original untransformed GS 115 strainwas also grown and taken through the same procedure. As a positivecontrol for methanol induction and secretion of a protein into themedia, a GS 115 strain containing wild-type HSA integrated into thePichia genome under control of the methanol-inducible promoter was alsogrown. The colonies expressed aldehyde-tagged-HSA and secreted it intothe media (see FIG. 14).

Example 9 Expressing and Purifying Aldehyde-Tagged-HSA from CHO Cells

24 μg of a DNA construct containing aldehyde-tagged-HSA in pcDNA3.1(pRW38) was transfected into CHO-K1 cells in Opti-MEM serum-free mediumusing Lipofectin transfection reagent in a 10 cm dish. After 5 h at 37°C., the Opti-MEM was removed and Ex-Cell 325 protein-free medium (+1%FBS+L-glut+Pen/Strep) was added. After 72 h at 37°, the media wascollected and cleared of debris. 10 mL Binding Buffer (20 mM Na₂PO₄, 500mM NaCl, 20 mM Imidazole, pH 7.5) and 200 μl of Ni-NTA resin was added.After incubating with rotation for 1 h at 4° C., the mixture was addedto a column and the flow-through fraction was collected. The resin waswashed with 4 mL Binding Buffer and then eluted 5 times with 500 μLElution Buffer (20 mM Na₂PO₄, 500 mM NaCl, 500 mM Imidazole, pH 7.5). 10μL of the media was run on an SDS-PAGE gel, and the gel was stained withCoomassie Blue (FIG. 15).

1.-33. (canceled)
 34. A method of producing a carrier protein-drugconjugate, the method comprising: (a) combining in a reaction mixture:(1) an aldehyde-tagged carrier protein comprising a heterologoussulfatase motif, wherein the heterologous sulfatase motif is less than13 amino acid residues and contains a sequence of the formula:X₁Z₁X₂Z₂X₃Z₃ wherein: Z₁ is a 2-formylglycine residue; Z₂ is a prolineor alanine residue; X₁ is present or absent and, when present, is anyamino acid, wherein X₁ is present when the sulfatase motif is at theN-terminus of the polypeptide; X₂ and X₃ are each independently anyamino acid; and Z₃ is a basic amino acid; and (2) a drug for conjugationto the carrier protein, wherein the drug comprises a reactive partnerfor an aldehyde of the carrier protein; wherein the drug is provided inthe reaction mixture in an amount sufficient to provide for a desireddrug to carrier protein ratio, said combining being under conditionssuitable to promote reaction between the aldehyde of the carrier proteinand the reactive partner of the drug to generate a carrier protein-drugconjugate; and (b) isolating the carrier protein-drug conjugate from thereaction mixture; and wherein the heterologous sulfatase motif of thecarrier protein-drug conjugate contains a sequence of the formula:X₁(FGly′)X₂Z₂X₃Z₃ where FGly′ is of the formula:

wherein: J¹ is the drug; each L¹ is independently selected fromalkylene, substituted alkylene, alkenylene, substituted alkenylene,alkynylene, substituted alkynylene, arylene, substituted arylene,cycloalkylene, substituted cycloalkylene, heteroarylene, substitutedheteroarylene, heterocyclene, substituted heterocyclene, acyl, amido,acyloxy, urethanylene, thioester, sulfonyl, sulfonamide, sulfonyl ester,O, and NH; and n is a number selected from 1 to
 40. 35. The method ofclaim 34, wherein the carrier protein is folded prior to said combining.36. The method of claim 35, wherein the carrier protein presents thedrug on a solvent-accessible surface of the carrier protein-drugconjugate when the carrier protein is folded.
 37. The method of claim34, where in the carrier protein-drug conjugate comprises two or moreheterologous sulfatase motifs.
 38. The method of claim 37, where in thecarrier protein-drug conjugate comprises three or more heterologoussulfatase motifs.
 39. The method of claim 37, where in the heterologoussulfatase motifs are positioned in the carrier protein-drug conjugate atat least one of the N-terminus of the carrier protein, the C-terminus ofthe carrier protein, and a solvent-accessible loop of the carrierprotein.
 40. The method of claim 37, wherein the two or moreheterologous sulfatase motifs are provided as a concatamer and areseparated by a flexible linker.
 41. The method of claim 34, wherein thecarrier protein-drug conjugate has a drug to carrier protein ratio of2:1 or more.
 42. The method of claim 34, wherein the carrierprotein-drug conjugate has a drug to carrier protein ratio of 3:1 ormore.
 43. The method of claim 34, wherein the carrier protein-drugconjugate has a drug to carrier protein ratio of 4:1 or more.
 44. Themethod of claim 34, wherein the carrier protein-drug conjugate has adrug to carrier protein ratio of 5:1 or more.
 45. The method of claim34, wherein the carrier protein is albumin.
 46. The method of claim 34,wherein the covalently bound drug is a peptide drug.
 47. The method ofclaim 46, wherein the peptide drug is glucagon-like peptide 1 (GLP-1) ora biologically active variant thereof.
 48. The method of claim 46,wherein the peptide drug is calcitonin or a biologically active variantthereof.
 49. The method of claim 34, wherein the covalently bound drugis a small molecule drug.
 50. The method of claim 34, wherein Z₃ isarginine (R).
 51. The method of claim 34, wherein X₁, when present, isan aliphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid; and X₂, and X₃ are each independently an aliphaticamino acid, a sulfur-containing amino acid, or a polar, uncharged aminoacid.
 52. The method of claim 34, wherein the X₁, when present, is L, M,V, S or T.
 53. The method of claim 34, wherein X₂ and X₃ are eachindependently S, T, A, V, G, or C.
 54. The method of claim 34, whereinthe heterologous sulfatase motif is less than 12 amino acid residues.55. The method of claim 34, wherein the heterologous sulfatase motif isless than 11 amino acid residues.
 56. The method of claim 34, whereinthe heterologous sulfatase motif is less than 10 amino acid residues.57. The method of claim 34, wherein the heterologous sulfatase motif isless than 9 amino acid residues.
 58. The method of claim 34, wherein theheterologous sulfatase motif is less than 8 amino acid residues.
 59. Themethod of claim 34, wherein the heterologous sulfatase motif is lessthan 7 amino acid residues.